CONTRAST AGENTS FOR DETECTION OF ENZYME ACTIVITIES BASED ON MELANIN SYNTHESIS

We disclose a composition, comprising a compound, comprising a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, and a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity. We also disclose methods of tumor imaging, tumor detection, diagnosis, and treatment, including methods comprising administering, to a patient suffering from a tumor, the composition; and either surgically resecting the tumor or thermally ablating the tumor, wherein the surgical resection is guided at least in part by contrast imparted by, or the thermally ablating targets cells containing, the melanin, wherein the melanin is spontaneously synthesized in the tumor after administering the composition.

Skip to: Description  ·  Claims  · Patent History  ·  Patent History
Description
GOVERNMENT SUPPORT STATEMENT

This invention was made with government support under grant number EB028062 from the National Institute for Biomedical Imaging and Bioengineering of the National Institutes of Health of the United States of America. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of cancer treatment. More particularly, it concerns contrast agents for detection of enzyme activities, such as protease activities, based on melanin synthesis.

BACKGROUND OF THE INVENTION

Surgical resection is a well-known technique for the treatment of solid tumors. However, surgical resection is limited by the difficulty of distinguishing healthy tissue from tumors, especially small tumors.

Prior workers have developed optical surgical navigation using fluorescent contrast agents. The contrast agent is targeted to the tumor and fluorescence is induced. Fluorescent contrast agents can preferentially highlight tumors, especially many small tumors within normal tissue, thereby improving the detection of tumors during surgery.

However, optical surgical navigation using fluorescent contrast agents has several shortcomings. For example, firstly, fluorescence contrast agents must be guided to the tumor to be effective. As a second example, fluorescence imaging instruments must be introduced to the surgical arena. The fluorescence imaging instruments must be placed in proximity to the patient, which may hinder the ability of the surgical team to operate. To maximize the visible contrast of most fluorophores currently known, the operating room should be kept dark, which may further hinder the surgical team. Also, fluorophores, being unnatural, may impose a serious compliance and regulatory-approval burden.

Accordingly, it would be desirable to develop optical surgical navigation techniques using one or more of naturally-occurring and biocompatible contrast agents, contrast agents that are preferentially activated in tumors, and that impart optical contrast that requires little or no instrumentation to induce or detect. It would be desirable for such optical contrast agents to be detectable by known, non-fluorescence-based techniques and/or to provide optical contrast visible to the naked eye.

SUMMARY OF THE INVENTION

The following presents a simplified summary of the disclosure in order to provide a basic understanding of some aspects of the disclosure. This summary is not an exhaustive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the disclosure. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed later.

In one embodiment, the present disclosure relates to a composition, comprising: a compound, comprising: a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, and a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity.

In one embodiment, the present disclosure relates to a method, comprising: administering, to a patient suffering from a tumor, a composition as referred to above; and surgically resecting the tumor, wherein the surgical resection is guided at least in part by contrast imparted by the melanin, wherein the melanin is spontaneously synthesized in the tumor after administering the composition.

In one embodiment, the present disclosure relates to a method, comprising: administering, to a patient suffering from a tumor, a composition as referred to above; and thermally ablating the tumor.

In one embodiment, the present disclosure relates to a method, comprising: administering, to a patient suffering from a tumor, a composition as referred to above; and detecting the tumor with noninvasive imaging.

In one embodiment, the present disclosure relates to a kit, comprising a composition as described above; and instructions for use of the composition in a method comprising administering, to a patient suffering from a tumor, the composition; and surgically resecting and/or thermally ablating the tumor, wherein the surgical resection is guided at least in part by contrast imparted by, or the thermally ablating targets cells containing, melanin spontaneously synthesized by the tumor after administering the composition.

In one embodiment, the present disclosure relates to a composition, comprising: a compound, comprising: a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, a linker moiety covalently bonded to the melanin precursor, and a carbohydrate covalently bonded to the linker moiety, wherein the indirect link of the carbohydrate to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of hydrolase activity.

In one embodiment, the present disclosure relates to a method, comprising: inserting, into at least one cell of an organism, a reporter gene construct comprising a regulatory sequence of interest and a coding sequence for a hydrolase enzyme; administering, to the cell, a composition comprising a compound comprising a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, a linker moiety covalently bonded to the melanin precursor, and a carbohydrate covalently bonded to the linker moiety, wherein the indirect link of the carbohydrate to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of hydrolase activity; and observing melanin in the cell, wherein the melanin is spontaneously synthesized in the cell after administering the composition.

The compositions disclosed herein may be used in methods and kits disclosed herein to provide optical surgical navigation using one or more of naturally-occurring and biocompatible contrast agents, contrast agents that are preferentially activated in tumors, and that impart optical contrast that requires little instrumentation to induce. The compositions may provide optical contrast to the naked eye.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 schematically depicts biochemical reactions that lead from naturally-occurring melanin precursors to formation of eumelanin and pheomelanin.

FIG. 2 schematically depicts biochemical reactions that lead from Agents 1-3 to formation of eumelanin and pheomelanin, in accordance with embodiments herein.

FIG. 3 schematically depicts an exemplary plan for synthesis of Agents 1-3, in accordance with embodiments herein.

FIG. 4 presents a flowchart of a first method in accordance with embodiments herein.

FIG. 5 presents a flowchart of a second method in accordance with embodiments herein.

FIG. 6 reports multispectral optoacoustic tomography (MSOT), also known as photoacoustic imaging (PAI), spectra of eumelanin and pheomelanin, normalized to 100% signal at 700 nm, in accordance with embodiments herein.

FIG. 7 shows models of melanins as contrast agents, in accordance with embodiments herein.

FIG. 8 presents a flowchart of a third method in accordance with embodiments herein.

FIG. 9 presents a flowchart of a fourth method in accordance with embodiments herein.

FIG. 10 schematically depicts biochemical reactions that lead from naturally-occurring melanin precursors to formation of eumelanin and pheomelanin.

FIG. 11 schematically depicts an exemplary plan for synthesis of Agent 6, in accordance with embodiments herein.

FIG. 12A presents multispectral optoacoustic (MSOT) imaging spectra of various concentrations of Agent 6 incubated with cathepsin B, as described in Example 1.

FIG. 12B presents absorbance spectra of the various concentrations of Agent 6 incubated with cathepsin B, as described in Example 1.

FIG. 12C presents MSOT signal-to-noise (SNR) ratios for the various concentrations of Agent 6, as described in Example 1.

FIG. 12D presents absorbance signal-to-noise (SNR) ratios for the various concentrations of Agent 6, as described in Example 1.

While the subject matter disclosed herein is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description herein of specific embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Moreover, the stylized depictions illustrated in the drawings are not drawn to any absolute scale.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various illustrative embodiments of the disclosure are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related, regulatory, and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

The present subject matter will now be described with reference to the attached figures. Various structures, systems, and devices are schematically depicted in the drawings for purposes of explanation only and so as to not obscure the present disclosure with details that are well known to those skilled in the art. Nevertheless, the attached drawings are included to describe and explain illustrative examples of the present disclosure. The words and phrases used herein should be understood and interpreted to have a meaning consistent with the understanding of those words and phrases by those skilled in the relevant art. No special definition of a term or phrase, i.e., a definition that is different from the ordinary and customary meaning as understood by those skilled in the art, is intended to be implied by consistent usage of the term or phrase herein. To the extent that a term or phrase is intended to have a special meaning, i.e., a meaning other than that understood by skilled artisans, such a special definition will be expressly set forth in the specification in a definitional manner that directly and unequivocally provides the special definition for the term or phrase.

As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.

Throughout this application, any given numerical value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists between study subjects or healthcare practitioners.

In one embodiment, the present disclosure relates to a composition, comprising a compound. The compound comprises a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo. The compound also comprises a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity.

A “melanin precursor” as used herein refers to any compound that spontaneously synthesizes a melanin when in free form in vivo. As is known to the person of ordinary skill in the art, “melanin” is a term for several polymeric pigments common in humans, other animals, plants, and microorganism. One common melanin in humans is eumelanin, which typically comprises cross-linked 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) polymers. Depending on the proportions of DHI and DHICA in a eumelanin, eumelanin may be further characterized as brown eumelanin and black eumelanin. A second common melanin in humans is pheomelanin, which differs from eumelanin by typically incorporating benzothiazine and benzothiazole units. At naturally-occurring concentrations in humans, pheomelanins generally impart yellow and red tints to various body features.

“Spontaneous” and grammatical variations thereof, when used herein to refer to a biochemical reaction, means that the biochemical reaction takes place without requiring enzymatic activity. For example, spontaneous formation of a melanin from a melanin precursor forms and/or adds onto a melanin without requiring enzymatic activity.

FIG. 1 schematically depicts biochemical reactions that lead from naturally-occurring melanin precursors to formation of eumelanin and pheomelanin. Though not intended to be exhaustive of all such reactions, FIG. 1 shows that tyrosinase catalyzes the oxidation of tyrosine (upper left) and L-DOPA (also known as levodopa and 1-3,4-dihydroxyphenylalanine; upper right) to yield dopaquinone, that spontaneously converts to leucodopachrome and cysteinyldopa. Leucodopachrome generally, but not exclusively, spontaneously polymerizes to form eumelanin. Cysteinyldopa generally, but not exclusively, spontaneously polymerizes to form pheomelanin.

Accordingly, in one embodiment, the melanin precursor is selected from the group consisting of leucodopachrome, decarboxylated leucodopachrome, and cysteinyldopa.

FIG. 10 schematically depicts other biochemical reactions that lead from naturally-occurring melanin precursors to formation of eumelanin and pheomelanin. Though not intended to be exhaustive of all such reactions, FIG. 10 shows that the formation of eumelanin may be summarized (A) as the oxidation of tyrosine or L-DOPA by tyrosinase to form dopaquinone, followed by spontaneous polymerization to form eumelanin. Pathway (B) shows in more detail that tyrosine is converted to L-DOPA, and L-DOPA to dopaquinone, by tyrosinase, followed by multiple steps to form an indole-quinone, which may then spontaneously polymerized to form eumelanin. Eumelanin can be detected with Multispectral Optoacoustic Tomography/Photoacoustic Imaging (PAL conceptually represented as white circles on black squares).

Accordingly, other melanin precursors, such as DHI and DHICA, among others, may be used in the compound.

As stated above, the compound comprises a melanin precursor and a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity.

A peptide has a standard meaning in the art and need not be discussed in detail. The peptide may comprise any number of amino acids, and hence, encompasses “oligopeptides,” “polypeptides,” and “proteins,” as those terms are commonly used in the art. Each of the amino acids in the peptide may be independently selected from any known amino acid molecule, i.e., may be selected without being bound by the selection of any amino acid incorporated into the peptide at any other position. The peptide may be synthesized by chemical techniques or purified from a naturally-occurring source.

The chemical formula of the peptide may be chosen without consideration of biochemical activities of the free peptide. However, in embodiments, the peptide may have a desirable biochemical activity that it may perform after cleavage of the peptide from the compound by a protease in a tumor. In one embodiment, the peptide may have an anti-tumor activity. For example, the peptide may be cyclic arginylglycylaspartic acid (RGD). Peptides with anti-tumor activities are known and their inclusion into the compound may be performed as a routine matter by the person of ordinary skill in the art having the benefit of the present disclosure.

Alternatively or in addition, the peptide may be linked, at a location other than the covalent bond from the peptide to the linker or the melanin precursor, with a therapeutic agent. In one embodiment, the therapeutic agent is not active until the peptide is cleaved by tumor protease. For one, non-limiting, example, a therapeutic agent may have a poor ability to enter cells when attached to the peptide-melanin precursor compound, but may have an improved ability to enter cells after the peptide is cleaved from the melanin precursor.

By “directly covalently bonded to” regarding the peptide and the melanin precursor is meant that the N-terminal nitrogen, the C-terminal carbon, or an atom on a side chain of an amino acid of the peptide is bonded to an atom of the melanin precursor. The atom of the melanin precursor may, but need not necessarily, be incorporated into the melanin. By “indirectly linked” regarding the melanin precursor and the peptide is meant that the N-terminal nitrogen, the C-terminal carbon, or an atom on a side chain of an amino acid of the peptide is bonded to a first atom of a linker moiety that is not the melanin precursor, and an atom of the melanin precursor is also bonded to a second atom of the linker moiety. Typically, but not necessarily, the first atom and the second atom are different atoms.

Any linker moiety known to the person of ordinary skill in the art as being suitable for use in compounds administered in vivo can be used. In one embodiment, the linker is capable of spontaneous disassembly from the melanin precursor after the peptide is removed.

In one embodiment, the indirect link comprises a p-hydroxybenzyl moiety. In another embodiment, the indirect link may comprise a 4-aminobenzoic acid (PABA) moiety.

Regardless of the type of link, wherein the direct covalent bond or indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity.

Protease activity, also known as peptidase activity, is an enzyme-catalyzed process by which peptide bonds are hydrolyzed. Typically, protease activity in healthy human and animal cells is sufficiently low that, in the event that the compound is introduced into such cells at reasonable concentrations, the rate of cleavage of the peptide-melanin precursor or peptide-linker bond is slower than the rate of spontaneous formation of a melanin from the melanin precursor, thereby allowing other naturally-occurring biochemical reactions to clear the melanin precursor from the cells before it can form a melanin. The “absence of protease activity” may be determined observationally simply from a lack of formation of pigment in cells to which the compound is introduced.

Many tumors, on the other hand, are characterized by significant protease activity within their cells. For example, MDA-MB-231 mammary carcinoma typically presents with high expression and secretion of cathepsin B and uPA, both of which are proteases. MDF7-TamR mammary carcinoma typically presents with high expression and secretion of uPA.

Accordingly, if the compound is introduced to cells of tumor characterized by significant protease activity, protease(s) cleave the peptide, leaving either the melanin precursor, which spontaneously forms a melanin in vivo, or the melanin precursor attached to a linker moiety. If the linker moiety is chosen to be a spontaneously-disassembling linker moiety (e.g., a p-hydroxybenzyl moiety), after disassembly of the linker moiety, a melanin precursor remains, which spontaneously forms a melanin in vivo.

FIG. 2 schematically depicts biochemical reactions discussed above in more detail. Beginning with one of three compounds, Agents 1-3, tumor protease(s) cleave the peptide, leaving either a melanin precursor-linker moiety molecule (produced from Agents 1-2) or cysteinyldopa (produced from Agent 3). After the linker moiety spontaneously disassembles, it yields leucodopachrome or cysteinyldopa (derived from Agents 1 and 2, respectively). The leucodopachrome and cysteinyldopa derived from Agents 1-3 then spontaneously form eumelanin and pheomelanin, respectively, in vivo.

FIG. 10 supplements FIG. 2 by showing that leucodopachrome spontaneously decarboxylates as part of its spontaneous polymerization to yield eumelanin. Accordingly, the melanin precursor may be a decarboxylated leucodopachrome, such as the melanin precursor identified below as Agent 6.

In one embodiment, the compound is selected from the group consisting of Agent 1, Agent 2, Agent 3, and Agent 6:

wherein n is greater than or equal to 1 and each R may be any organic moiety.

Agents 1-4 are exemplary compounds comprising the melanin precursor and the peptide. The person of ordinary skill in the art having the benefit of the present disclosure will be able to synthesize and use other molecules meeting the limitations of the disclosed compounds as a routine mater and would be in the spirit and scope of the embodiments disclosed herein.

The compound may be synthesized using any appropriate technique known to the person of ordinary skill in the art. FIG. 3 schematically depicts an exemplary and non-limiting plan for synthesis of Agents 1-3. Agent 1 can be synthesized using steps A-G. Agent 2 can be synthesized using steps A,H,I,C,E,F,G. Agent 3 can be synthesized using steps A,H,I,C,E,G. Agents that detect cathepsin B and urokinase plasminogen activator (uPA) can use HRYR and ASGK peptides. A protected peptide with a C-terminal hydroxybenzyl group is a reactant in step F, and a protected dipeptide with a disulfide bridge is a reactant in step H, which are synthesized using standard peptide synthesis methods.95-106 All reactions are high yield, except step H. However, step H uses inexpensive reagents and is an initial step, so that a large amount can still be obtained from this step.

Reagents and reaction conditions for steps A-H can be as follows:

A) AcCl, AlCl3, PhNO2, 100° C.

B) SOCl2, MeOH, 0° C. to r.t., then picolinic acid, EDC, HOBt, DIPEA, DCM

C) TBDMSCl, imidazole, DMF, r.t.

D) Pd(OAc2), PhI(Ac2O), toluene, 60° C., Ar

E) mCPBA, CH2Cl2, r.t., then NH3MeOH, r.t.

F) K2CO3, NMP, r.t., then MsCl, DIPEA, NMP, −10° C. to r.t.

G) KOH, H2O, MeOH, reflux

H) HBr, 100° C., 6 h

I) SOCl2, MeOH, 0° C. to r.t., then Ac2O, pyridine, r.t.

FIG. 11 shows an exemplary and non-limiting synthesis scheme for the formation of Agent 6. Compound 1. 3,4-dihydroxyindole (200 mg, 1.45 mmol) was dissolved in dry DMF (4 mL), and potassium carbonate (800 mg, 5.79 mmol) and benzyl bromide (0.69 mL, 5.79 mmol) were added. The reaction was heated to 65° C. and stirred overnight. The solution was then diluted with ethyl acetate and washed with water then brine. The organic layer was dried over MgSO4 and evaporated under reduced pressure. The resulting solid was purified by column chromatography over silica gel eluting with 20% ethyl acetate in hexanes to afford the desired product as a white solid (446 mg, 97% yield).

Compound 2. Compound 1 (524 mg, 1.65 mmol), nitromethane (0.53 mL, 9.88 mmol), ammonium acetate (508 mg, 6.58 mmol), and glacial acetic acid (6 mL) was combined, heated to reflux, and stirred for 1 hour. After cooling to room temperature, most of the acetic acid was removed under reduced pressure, and DCM was added. The solution was washed with a saturated solution of sodium carbonate followed by water. The organic layer was then dried over MgSO4 and evaporated under reduced pressure. The resulting yellow solid was pure by TLC, so it was used without further purification (548 mg, 92% yield).

Compound 3. Compound 2 (548 mg, 1.52 mmol) was dissolved in glacial acetic acid (8 mL), and nitric acid (0.91 mL) was added dropwise. The reaction was heated to 40° C. and stirred for 2 hours. The reaction mixture was then poured into ice, and a precipitate formed. The solid was collected, dissolved in DCM, and washed with a saturated solution of sodium carbonate. The organic layer was then dried over MgSO4 and evaporated under reduced pressure. The resulting solid was dissolved in a minimal amount of DCM, and methanol was added to crash out the desired product as a yellow solid (411 mg, 67% yield).

Compound 4. TFA (8 mL) was added to compound 3 (200 mg, 0.492 mmol), and the reaction mixture was heated to reflux for 3 hours. The solution was then cooled in an ice bath for 30 mins, and the solid was filtered out and washed with diethyl ether. The filtrate was evaporated under reduced pressure, and the resulting yellow solid was used without further purification (187 mg, quantitative yield).

Compound 5. Compound 4 (13 mg, 0.058 mmol) and Ac-Phe-Lys(Boc)-PABA (32 mg, 0.058 mmol) was dissolved in dry THF (1.5 mL) and cooled to −20° C. Triphenylphosphine (23 mg, 0.088 mmol) was added, and the reaction was stirred for 15 mins. DEAD (0.014 mL, 0.088 mmol) was then added dropwise and stirred for 1 hour at −20° C. before being allowed to warm to room temperature. After stirring overnight at room temperature, the reaction was diluted with ethyl acetate and washed with water followed by brine. The organic layer was dried over MgSO4 and evaporated under reduced pressure. The resulting solid was purified by column chromatography over silica gel eluting with 5% methanol in DCM to afford the desired product as a white solid (18 mg, 78% yield).

Compound 6. Compound 5 (18 mg, 0.045 mmol) was dissolved in dry ethanol (1 mL). ZnSO4·H2O (177 mg, 0.984 mmol) and Na2S2O4 (321 mg, 1.845 mmol) were dissolved in de-gassed 0.1 M PBS, pH=4 (1 mL) and added to the solution of compound 5. The reaction mixture was heated to 50° C. and stirred for 3 hours. Most of the ethanol was then removed under reduced pressure and extracted with ethyl acetate three times. The organic layers were combined, dried over MgSO4, and evaporated under reduced pressure. The resulting solid was purified by column chromatography over silica gel eluting with 20% ethyl acetate in hexanes to afford the desired product as a brown solid (14 mg, 68% yield).

In addition to the compound, the composition may further comprise one or more pharmaceutically-acceptable compounds, such as carriers (e.g., solvents, inert materials used in compounding tablets for oral ingestion, wall materials degradable in the patient's body for use in capsules for oral or other routes of administration), buffers, preservatives, adjuvants, surfactants, diluents (e.g. saline or dextrose) or the like. Such particular other compounds may be routinely selected by the person of ordinary skill in the art having the benefit of the present disclosure.

In one embodiment, the composition further comprises a pharmaceutically-acceptable carrier.

The composition may be formulated in a solid form, such as a soluble powder, a tablet, a caplet, or a capsule, among others; in a liquid form, such as a solution or slurry in an aqueous solvent, a hydrophilic solvent, or a hydrophobic solvent; or in an aerosol form, among others. The particular formulation and the concentration of the compound in the composition may be chosen by the person of ordinary skill in the art having the benefit of the present disclosure as a routine matter.

Turning to FIG. 4, a flowchart of a method 400 in accordance with embodiments herein, is presented. The method 400 comprises administering (at 410), to a patient suffering from a tumor, a composition comprising a compound comprising a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo. The composition also comprises a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity.

The composition, the compound, the melanin precursor, the peptide, and the linker moiety providing the indirect linkage, if any, may be as described above.

The patient may be any mammal suffering from the cancer. In one embodiment, the patient is a human being.

In embodiments, the present method may be performed in a veterinary context. That is, the patient may be any non-human mammal suffering from a cancer. The non-human mammal may be a research animal, a pet, livestock, a working animal, a racing animal (e.g., a horse, a dog, a camel, etc.), an animal at stud (e.g., a bull, a retired racing stallion, etc.), or any other non-human mammal for which it is desired to treat its cancer.

For convenience, the description will typically refer to human patients. However, the person of ordinary skill in the art having the benefit of the present disclosure will readily be able to adapt the teachings of the present disclosure to a veterinary context.

By “suffering from a tumor” is meant that the tumor is detectable in the patient's body using any diagnostic technique presently known or to be discovered. The tumor may be benign, premalignant, or malignant. The tumor need not have been directly detected, e.g., a prostate cancer tumor may be detected from elevated levels of prostate-specific antigen in the patient's blood. “Suffering” does not require the patient to be in pain from or have any naturally-perceptible symptoms of the tumor. Generally, as is known, the earlier a tumor can be treated, including before the patient notices pain or any other symptoms, the greater the chances of remission.

The present method may be used to treat any tumor of any type of cancer. Desirably, the tumor is a solid tumor comprising cells that express high levels of protease. “High levels” as used herein refers to an increase in protease activity over the level prevailing in non-tumor tissue of the type from which the tumor originated. In one embodiment, the increase may be at least 75%. In another embodiment, the increase may be at least 50%. In yet another embodiment, the increase may be at least 25%. In still another embodiment, the increase may be at least 10%. Also desirably, the tumor is one that is known or reasonably expected, by the person of ordinary skill in the art having the benefit of the present disclosure, to be treatable by surgical resection or heat therapy.

Almost all malignant and metastatic tumors are expected to have upregulated extracellular proteases.11,12 As a fundamental concept in tumor biology, a proteolytic cascade is required to degrade the extracellular matrix and cellular stroma to allow for tumor cell invasion (malignancy) and escape (metastasis). This fundamental concept has spurred the development of protease-activated fluorescent dyes for optical guided surgery

Desirably, the tumor is of a cancer type and in an organ with essentially zero naturally-occurring melanin. Typically, melanins are only present at high concentrations in primary and metastatic melanoma, hair, and skin, and at low concentrations in brain and spine. We expect other types of tumors and tumors in other organs to be amenable to the methods disclosed herein.

In one embodiment, the tumor is of a cancer selected from the group consisting of breast cancer, head-and-neck cancer, pancreatic cancer, ovarian cancer, and thyroid cancer.

In one particular embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is head-and-neck cancer. In an embodiment, the cancer is breast cancer. In an additional embodiment, the cancer is ovarian cancer. In yet an additional embodiment, the cancer is thyroid cancer.

The composition may be administered 410 to the patient by any route and remain within the spirit and scope of the embodiments described herein. Such routes may be characterized as systemic or local. Systemic routes include oral, nasal, buccal, and intravenous injection routes, among others. Local routes include subcutaneous, intramuscular, intraorganal, and intratumoral injection, and catheterized and endoscopic routes, among others.

In one embodiment, administering 410 the composition comprises intravenous injection of the composition as an aqueous solution into the patient. Other systemic delivery techniques for administering 410 the composition are known to the person of ordinary skill in the art having the benefit of the present disclosure and may be selected by such person as a routine matter.

In the method 400, administering 410 the composition may be performed in a single dose or a plurality of doses. In one embodiment, a single dose is administered 410. The total dosage may provide the compound in a concentration from about 0.1 μmol/kg body weight to 1 mmol/kg body weight. In one embodiment, the total dosage may provide the compound in a concentration from about 0.5 μmol/kg body weight to 60 μmol/kg body weight.

The method 400 typically requires some time for the compound to be taken up by the tumor, followed by allowing (at 412) protease in the tumor to cleave the peptide from the melanin precursor or a compound containing the melanin precursor and a linker moiety, allowing the linker moiety (if present) to spontaneously disassemble, and allowing (at 414) the melanin precursor to spontaneously form melanin in the tumor. The total time required from administering 410 until a desirably high level of melanin has formed in the tumor may be from 0.1 hr to 72 hr. In one embodiment, the required time may be from about 0.25 hr to about 24 hr.

If desired, the method 400 may comprise detecting (at 416) the spontaneously-synthesized melanin in the tumor. The detecting (at 416) may be performed before, during, after, or in lieu of surgical resection (at 420), discussed below. The detecting (at 416) may comprise inspection by the naked eye or qualitative or quantitative, real-time or time-delayed, and/or still or video image observation by multispectral optoacoustic tomography (MSOT), also known as photoacoustic imaging (PAI).

The method 400 may further comprise surgically resecting (at 420) the tumor, wherein the surgical resection is guided at least in part by contrast imparted by the melanin, wherein the melanin is spontaneously synthesized in the tumor after administering the composition.

The melanin may impart contrast in a number of ways. For example, melanins can be detected using MSOT/PAI. Both eumelanin and pheomelanin have strong signals across a broad spectral range that generates strong detection with MSOT (FIG. 6).

A small animal MSOT instrument (InVision) can rapidly acquire multispectral images of an entire mouse torso at 150 μm in-plane resolution and 1 mm slice thickness.32,33 The strong laser of this instrument can image the entire transaxial≤3 cm diameter of the mouse torso, overcoming the limited depth of view of most other optoacoustic imaging instruments.

A clinical MSOT instrument can image tumors and other tissues, including in three dimensions. For example, the Acuity clinical MSOT instrument operates at a pulse repetition rate of 50 Hz to accommodate the movement of the transceiver wand. The RapidSCAN™ data acquisition system has 384 channels that can sample as fast as 40 MS/s. The three-dimensional (3D) detector covers a 180° angle with 384 detector elements to provide excellent sensitivity.

The laser for the clinical imaging system operates at ≤30 mJ with 4-10 nsec pulses to ensure clinical safety. These performance characteristics comply with ANSI Maximum Permissible Energy (MPE). The class 4 laser produces wavelengths from 680 to 980 nm in 1 nm increments, with almost-negligible<10 msec tuning at each wavelength.

The OPUS™ ultrasound imaging system has a broadband 2-8 MHz frequency range with synthetic aperture beamforming and spatial compounding. Prototypes for this system have shown sentinel lymph node detection in the clinic at 2 cm depth of field, and at a 5 cm depth of field with gentle pressing on the body, and with a very sensitive detection sensitivity of 4 cells with physiological melanin content. Melanin concentrations under 100 nM may be detectable by MSOT in vivo.

A depth of view of 3 cm has been shown to be sufficient to image patients with breast cancel34-36 and other pathologies37,38. Moreover, MSOT is being developed for intrasurgical imaging. Our technology can be used to localize protease-active tumors as deep as 3 cm from tissue surfaces that are exposed during surgery, further improving surgical resection of tumors. “Digging” for tumors below the exposed tissue surface is one of the greatest problems with fluorescence guided surgery, which can be addressed with MSOT guided surgery.

Another way melanin may impart contrast is by color in the visible spectrum of light. The administering 410 causes protease-active tumors to accumulate melanins and become black. Simple visual inspection without fluorescence imaging instrumentation can then detect these black tumors against the beige-to-red background of normal tissues. FIG. 7 presents images of mouse tissues on which were placed droplets (˜1 μM) eumelanin or pheomelanin. FIG. 7 proves the concept that accumulation of melanins in tumors can provide optical contrast to the naked eye.

By use of MSOT, visual inspection, or both, a surgeon may be guided when surgically resecting 420 more easily and efficiently than by use of prior surgical imaging techniques, such as fluorescence. This guidance may allow the surgeon to remove more of the tumor without excessive removal of healthy tissues, and/or provide one or more other advantages.

As is known in the art, multiple treatment modalities are often used to increase the likelihood of remission of a cancer. Accordingly, in one embodiment, the method 400 may further comprise administering (at 430), to the patient, a cancer treatment modality other than the surgical resection. Administering 430 the cancer treatment modality other than the surgical resection may be targeted against the same cancer as the resected tumor, against metastases thereof, against a primary tumor or metastases of a tumor other than the surgically resected tumor, or two or more thereof.

A wide variety of cancer treatment modalities other than surgical resection are known to the person of ordinary skill in the art and need not be described in detail here. By way of example, in one embodiment, the cancer treatment modality other than the surgical resection is selected from the group consisting of radiation, chemotherapy, immunotherapy, checkpoint inhibitor therapy, oncolytic virus therapy, peptide therapy (e.g., cyclic RGD), thermal therapy (e.g., thermal ablation by the delivery and absorption of heat, light energy, radio frequency energy, and/or microwave energy, and/or cryotherapy), and two or more thereof.

Regardless of the particular cancer treatment modality other than surgical resection, if one or more is/are administered 430, the administering 430 may be performed before, after, or simultaneously with the surgically resecting 420. Particular relative and absolute timing of surgically resecting 420 and administering 430 the other cancer treatment modality will be a routine matter for the person of ordinary skill in the art having the benefit of the present disclosure.

FIG. 5 presents a flowchart of a method 500 in accordance with embodiments of the present disclosure. The method 500 comprises administering (at 510), to a patient suffering from a tumor, a composition comprising a compound comprising a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, and a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity. Administering (at 510) may be performed essentially as administering (block 410) in the method 400 shown in FIG. 4 and need not be described further. Allowing (at 512) protease in the tumor to cleave the peptide, allowing (at 514) the precursor to spontaneously form melanin in the tumor, and detecting (at 516) melanin in the tumor may also be performed as described above regarding allowing (block 412), allowing (block 414), and detecting (block 416) in the method 400 shown in FIG. 4.

The method 500 also comprises thermally ablating (at 520) the tumor, wherein the thermal ablation targets cells containing melanin, wherein the melanin is spontaneously synthesized in the tumor after administering the composition. In one embodiment, the thermally ablating (at 520) comprises delivering energy, most commonly, light energy to the tumor after melanin has formed in the tumor. Though not to be bound by theory, the light energy is preferentially absorbed by the melanin, the light energy is then radiated as heat, and the radiated heat may kill or impair cells of the tumor. “Light energy” encompasses all infrared, visible, and ultraviolet wavelengths, and is not limited herein to light at visible wavelengths.

The amount and wavelength of light or other energy, the duration of delivery of the light or other energy, and other parameters of thermally ablating (at 520) the tumor, may be routinely selected by the person of ordinary skill in the art having the benefit of the present disclosure.

To thermally ablate (at 520), the energy may be delivered through the skin, if the tumor is at or near the skin. If the tumor is at or near a body structure through which an energy source may be introduced endoscopically, e.g., the nose, the mouth, the trachea, the bronchi, the gastrointestinal tract, the arterial vasculature, the urethra, or the vagina, the energy may be delivered endoscopically. If the tumor is not in proximity to the skin or a body structure amenable to endoscopy, or if intervening structures preclude delivery of energy through the skin, the thermally ablating (at 520) may be performed after exposing the tumor to the ambient environment, typically by surgery.

The method 500 may, in embodiments, further comprise administering 530, to the patient, a cancer treatment modality other than the thermal ablation. Examples of such cancer treatment modalities were discussed with reference to administering 430 in method 400 shown in FIG. 4 and surgically resecting 420 in method 400.

Specifically, in one embodiment of administering 530, the cancer treatment modality other than the thermal ablation is selected from the group consisting of radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitor therapy, oncolytic virus therapy, peptide therapy, cryotherapy, RFA, microwave ablation, surgical resection, and two or more thereof.

In one embodiment, the methods 400 and 500 may be co-performed, i.e., the administering 430 in method 400 may comprise thermal ablation 520, and/or administering 530 in method 500 may comprise surgically resecting 420 the tumor. The combination of surgically resecting 420 and thermally ablating 520 may further increase the chances of remission: thermally ablating 520 might kill tumor cells that happen to elude the surgeon's perception when she or he surgically resects 420 the tumor, and surgical resection 420 might remove the main body of tumors that are too large for thermal ablation 520 to kill in a single procedure.

Turning to FIG. 9, in one embodiment, the present disclosure relates to a method 900. The method 900 comprises treating (at 910) a patient suffering from a tumor at a first location in the patient's body. Treating (at 910) may be by any known technique, including, but not necessarily limited to, radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitor therapy, oncolytic virus therapy, peptide therapy, cryotherapy, RFA, microwave ablation, thermal ablation, surgical resection, and two or more thereof. The treating (at 910) may include surgical resection guided by contrast imparted by compositions and methods of the present disclosure and/or thermal ablation along the lines of the methods of the present disclosure.

The method 900 further comprises administering (at 920), to the patient, a composition comprising a compound comprising a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo. The composition also comprises a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity.

Administering (at 920) may be performed using any techniques described above. The composition administered (at 920) may be as described above.

After administering (at 920), the fate of the composition will vary depending on the outcome of treating (at 910). If treating (at 910) were fully successful, and the tumor disappeared, then normal tissues at the first location in the patient's body, which are expected to lack protease activity, would not cleave the peptide from the melanin precursor, and the melanin precursor would substantially be prevented from forming melanin. If treating (at 910) were partially successful, and the tumor reduced in size, then the tumor would be expected to produce melanin after tumor proteases cleave the peptide from the melanin precursor (at 922) and the melanin precursor spontaneously forms melanin (at 924). If treating (at 910) were unsuccessful, and the tumor increased in size, then the tumor would also be expected to produce melanin.

Accordingly, the method 900 further comprises detecting (at 930) melanin at or near the first location. The detection (at 930) may be the naked eye, or may be by use of an optical imaging apparatus, such as MSOT equipment as discussed above. The detection may be qualitative or quantitative, with the latter potentially including measuring or calculating the size or volume of the tumor and/or the absorption intensity of the melanin spontaneously synthesized in the tumor.

The method 900 also comprises determining (at 940) the patient's tumor status in response to the detecting (at 930). Determining (at 940) may, but need not, comprise comparing the results of the detecting (at 930) with a baseline detection that may have been provided by administering similarly to the administering (at 920) and detecting similarly to the detecting (at 930) prior to the treating (at 910).

To continue the qualitative examples presented above, if treating (at 910) were fully successful, then detecting (at 930) would be expected to detect essentially zero melanin at the first location in the patient's body, and determining (at 930) may involve indicating, to the physician and/or the patient, that the tumor has essentially disappeared. If treating (at 910) were partially successful, then detecting (at 930) would be expected to detect the new, reduced size of the tumor, and determining (at 930) may involve indicating, to the physician, the size reduction in qualitative or quantitative terms, from which the physician may apply his or her skill to determine whether the treatment performed (at 910) should be repeated, modified, continued, discontinued, or replaced by another treatment of the same or different modality. If treating (at 910) were unsuccessful, then detecting (at 930) would be expected to detect the tumor's unchanged or increased size, and determining (at 930) may involve indicating, to the physician, the size increase in qualitative or quantitative terms, from which the physician may apply his or her skill to determine whether the treatment performed (at 910) should be repeated, modified, continued, discontinued, or replaced by another treatment of the same or different modality, or if palliative or hospice care is in the patient's best interest.

Determining (at 940) may be automated, particularly if the detecting (at 930) yields quantifiable data relating to tumor size, tumor volume, and/or melanin absorption intensity (which is reasonably expected to be indicative of protease activity) and/or if a baseline detection was performed prior to treating (at 910). Automated determination may enhance reproducibility and consistency of tumor status determinations and may allow use of data sets and algorithms that, if used by a person mentally or with the aid of pen and paper, would be prohibitive of time and attention, possibly even requiring months or years to calculate, thereby rendering the results of such hypothetical manual determination out-of-date and not clinically useful.

In another embodiment, the present disclosure relates to a method, comprising: administering, to a patient suffering from a tumor, a composition as referred to above; and detecting the tumor with noninvasive imaging.

In this embodiment, the administering may be as describe above, and will be expected to lead to significant amounts of melanin formation in tumors relative to healthy tissue.

Depending on the location of the tumor, detecting with noninvasive imaging may comprise visual inspection of the patient's skin, visual inspection through an endoscopic device introduced into the patient's body, or MSOT, among other techniques known in the art or to be developed.

By detecting the tumor with noninvasive imaging, the tumor may be diagnosed and/or monitored with greater accuracy, greater speed, and/or less patient discomfort. Detecting the tumor with noninvasive imaging may allow the physician to better plan a surgical resection of the tumor.

In one embodiment, the present disclosure relates to a “kit,” wherein the kit comprising a composition as described above; and instructions for use of the composition in a method comprising administering, to a patient suffering from a tumor, the composition; and surgically resecting and/or thermally ablating the tumor, wherein the surgical resection is guided at least in part by optical contrast imparted by, or the thermal ablation targets cells containing, melanin spontaneously synthesized by the tumor after administering the composition.

A “kit,” as used herein, refers to a package containing the composition, and instructions of any form that are provided in connection with the composition in a manner such that a clinical professional will clearly recognize that the instructions are to be associated with the composition.

“Instructions” typically involve written text or graphics on or associated with packaging of compositions of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner. Written text or graphics may include a website URL or a QR code encoding a website URL, where other instructions or supplemental information may be provided in electronic form.

The kit may contain one or more containers, which can contain the composition or a component thereof. The kits also may contain instructions for mixing, diluting, or administering the composition. The kits also can include other containers with one or more solvents, surfactants, preservatives, and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting, or administering the composition to the patient in need of such treatment.

The composition may be provided in any suitable form, for example, as a liquid solution or as a dried material. When the composition provided is a dry material, the material may be reconstituted by the addition of solvent, which may also be provided by the kit. In embodiments where liquid forms of the composition are used, the liquid form may be concentrated or ready to use.

The kit, in one embodiment, may comprise a carrier being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like.

The composition is described above.

The methods are as described above.

In one embodiment, the present disclosure relates to a composition, comprising: a compound, comprising: a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, a linker moiety covalently bonded to the melanin precursor, and a carbohydrate covalently bonded to the linker moiety, wherein the indirect link of the carbohydrate to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of hydrolase activity.

The melanin precursor and the linker moiety may be as described above.

By “carbohydrate” is meant any compound from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Desirably, the carbohydrate may be chosen to be a substrate of a particular hydrolase enzyme as described below.

In one embodiment, the compound is selected from the group consisting of Agent 4:

In Agents 4-5, R3, R4, and R5 may independently be any organic moiety. In one embodiment, R3 is —H, R4 is —OH, and R5 is —CH3OH. In another embodiment, R3 is —OH, R4 is —H, and R5 is —COO−.

FIG. 8 presents a flowchart of a method 800 in accordance with embodiments of the present disclosure. The method 800 comprises inserting (at 810), into at least one cell of an organism, a reporter gene construct comprising a regulatory sequence of interest and a coding sequence for a hydrolase enzyme.

Reporter gene constructs are well known in the molecular biology arts. Typically, the reporter gene construct is a portion of a DNA molecule comprising the regulatory sequence, e.g., the promoter, of a gene of interest for which it is desired to investigate the expression in a cell of interest under one or more conditions. The coding sequence for the hydrolase enzyme may be introduced into the DNA molecule at a location and orientation under control of the regulatory sequence of interest using techniques that are long established in the molecular biology arts and need not be described in detail.

The DNA molecule may also comprise one or more other coding sequences under the control of one or more other regulatory sequences. Such other coding sequences may assist in the handling, processing, transfection, genomic incorporation, or the like of the DNA molecule and/or the reporter gene construct.

The DNA molecule may be selected as a routine matter for many organisms. The cell may be a prokaryotic cell, a cell of a single-celled eukaryote, a plant cell, an animal cell, or a human cell, among others. Reporter gene constructs may be used in cells in vitro or in vivo.

Inserting 810 may comprise any techniques known to the person of ordinary skill in the art and need not be described in detail.

In one embodiment, the coding sequence for the hydrolase enzyme encodes a hydrolase enzyme selected from the group consisting of β-galactosidase (β-gal) and β-glucuronidase (β-gus).

The method 800 also comprises administering (at 820), to the cell, a composition comprising a compound comprising a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, a linker moiety covalently bonded to the melanin precursor, and a carbohydrate covalently bonded to the linker moiety, wherein the indirect link of the carbohydrate to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of hydrolase activity.

The composition may be as described above. In embodiments wherein the hydrolase enzyme is β-gal, the compound may be selected from the group consisting of Agents 4-5, wherein R3 is —H, R4 is —OH, and R5 is —CH3OH. In embodiments wherein the hydrolase enzyme is β-gus, the compound may be selected from the group consisting of Agents 4-5, wherein, R3 is —OH, R4 is —H, and R5 is —COO−.

Administering 820 to the cell may be directly to the cell or a culture medium containing the cell, if the method 800 is performed in vitro, or locally or systemically to an organism of which the cell is a part, if performed in vivo.

After the composition is administered 820, the compound may enter the cell or come in contact with one or more proteins on the cell surface. Though not to be bound by theory, hydrolase enzyme expressed by the reporter gene construct, either constitutively or as induced by one or more conditions, is expected to cleave the carbohydrate from the compound. The linker moiety is then expected to spontaneously disassemble, yielding the melanin precursor, which then is expected to spontaneously polymerize to form the melanin. If the hydrolase enzyme is not significantly expressed, the cleavage of the carbohydrate is not expected, and melanin will not be formed spontaneously from the melanin precursor. Accordingly, the accumulation or lack thereof of melanin in the cell indicates whether the regulatory sequence of interest is active or not active in the cell under the condition(s) tested by the experiment.

The method 800 also comprises observing (at 830) melanin in the cell, wherein the melanin is spontaneously synthesized in the cell after administering the composition. Observation 830 may be by any appropriate technique. In one embodiment, observation is by MSOT.

Observing 830 melanin in the cell does not imply that only one cell may be the subject of the method 800. A plurality of cells and/or a portion or the entirety of a multicellular organism may be the subject of the method 800.

In one embodiment, the method 800 may further comprise observing (at 815), prior to administering 820 the compound, melanin in the cell, wherein the melanin so observed is that which naturally occurs in the cell. Desirably, the observing 815 uses the same technique that is used in observing 830.

In this embodiment of the method 800, the observing 830 may comprise processing the data generated by observing 830 in view of the data generated by observing 815. This may comprise aligning images taken from observing 815 and observing 830 and subtracting the intensity of a pixel from the data generated by the observing 815 from the intensity of the corresponding pixel from the data generated by the observing 830, among other processing techniques that will be apparent to the person of ordinary skill in the art having the benefit of the present disclosure.

In one embodiment, the present disclosure relates to a kit, comprising a compound comprising a melanin precursor, a linker moiety, and a carbohydrate, and instructions for performing the method 800.

EXAMPLES Example 1

We incubated 2.22 mg/mL (5 μmol, 0.1 mL) of Agent 6 with 2 units of cathepsin B at pH 6.8 and at 37° C. overnight. Although cathepsin B is typically considered to be an endoprotease, this enzyme can work as an exoprotease at lower pH relative to physiological pH 7.4. Also, the extracellular tumor microenvironment is known to be acidic, so that pH 6.8 is a better representation of tumor conditions. The resulting solution was a light gray color, indicating that the reaction product contained melanin that was produced after Agent 6 was cleaved by the cathepsin B protease.

Other samples of were prepared at 1.66, 1.11, 0.84, and 0.55 mg/mL. These solutions were inserted into a customized phantom for a multi spectral optoacoustic imaging instrument (MSOT; also known as photoacoustic imaging) (InVision instrument, iThera Medical). FIG. 12A-FIG. 12D present MSOT or absorbance spectra data for the various concentrations.

The phantoms were imaged at 700-900 nm absorbance wavelengths in 2 nm steps. (FIG. 12A). The samples were also analyzed with a spectrofluorometer with absorbance through the same wavelength range (FIG. 12B), which matched the spectral profile and relative signal intensities of the MSOT spectra. The spectra were broad and featureless, which is expected for a melanin-based product. The signal to noise ratio (SNR) scaled with the concentration of the reactant, providing validation that the MSOT and absorbance signals were derived from the product and was not an artifact of the solution or imaging instrument (FIG. 12C, FIG. 12D).

Therefore, these preliminary results indicate that Agent 6 is cleaved by cathepsin B to create a melanin-based product that can be detected by MSOT.

REFERENCES

Detection of Protease Activity

  • 1. Mason H S. A classification of melanins. Ann New York Acad Sci 1948; 4:399-404. PMID: 18862181, no PMCID, no DOI.
  • 2. del Marmol V, Beermann F. Tyrosinase and related proteins in mammalian pigmentation. FEBS Lett 1996; 382:165-168. PMID: 8601447, no PMCID, no DOI.
  • 3. Grootendorst D J, Jose J, Wouters M W, van Boven H, Van der Hage J, Van Leeuwen D G, Steenbergen W, Manohar S, Ruers T J M. First experiences of photoacoustic imaging for detection of melanoma metastases in resected human lymph nodes. Lasers Surg Med 2012; 44:541-549. PMID: 22886491, no PMCID, DOI: 10.1002/1sm.22058
  • 4. Zhou Y, Li G, Zhu L, Li C, Cornelius L A, Wang L V. Handheld photoacoustic probe to detect both melanoma depth and volume at high speed in vivo. J Biophotonics 2015; 8:961-967. PMID: 25676898, PMCID: PMC4530093, DOI: 10.1002/jbio.201400143
  • 5. Stoffels I, Morscher S, Helfrich I, Hillen U, Leyh J, Burton N C, Sardella T C P, Claussen J, Poeppel T D, Bachmann H S, Roesch A, Briewank K, Schadendorf D, Gunzer M, Klode J. Metastatic status of sentinel lymph nodes in melanoma determined noninvasively with multispectral optoacoustic imaging. Sci Trans Med 2015; 7:317ra199. PMID: 26659573, no PMCID, DOI: 10.1126/scitranslmed.aad1278
  • 6. Schwarz M, Buehler A, Aguirre J, Ntziachristos V. Three-dimensional multispectral optoacoustic mesoscopy reveals melanin and blood oxygenation in human skin in vivo. J Biophotonics 2016; 9:55-60. PMID: 26530688, no PMCID, DOI: 10.1002/jbio.201500247
  • 7. Lavaud J, Henry M, Coll J L, Josserand V. Exploration of melanoma metastases in mice brains using endogenous contrast photoacoustic imaging. Int J Pharmaceutics 2017; 532:704-709. PMID: 28847669, no PMCID, DOI: 10.1016/j.ijpharm.2017.08.104
  • 8. Shah A, Delgado-Goni T, Casals Galobart T, Wantuch S, Jamin Y, Leach M O, Robinson S P, Bamber J, Beloueche-Babari M. Detecting human melanoma cell re-differentiation following BRAF or heat shock protein 90 inhibition using photoacoustic and magnetic resonance imaging. Sci Rep 2017; 7:8215. PMID: 28811486, PMCID: PMC5557970, DOI: 10.1038/s41598-017-07864-8
  • 9. Aggarwal N, Sloane B F. Cathepsin B: Multiple roles in cancer. Proteomics Clin App 2014; 8:427-437. PMID: 24677670, PMCID: PMC4205946, DOI: 10.1002/prca.201300105
  • 10. Stantibanez J F. Urokinase type plasminogen activator and the molecular mechanisms of its regulation in cancer. Protein Pept Lett 2017; 24:936-946. PMID: 28820062, no PMCID, DOI: 10.2174/0929866524666170818161132
  • 11. Mason S D, Joyce J A. Proteolytic networks in cancer. Trends Cell Biol 2011; 21:228-237. PMID: 21232958, PMCID: PMC3840715, DOI: 10.1016/j.tcb.2010.12.002
  • 12. McIntyre J O, Matrisian L M. Molecular imaging of proteolytic activity in cancer. J Cell Biochem 2003; 90:1087-1097. PMID: 14635184, no PMCID, DOI: 10.1002/jcb.10713
  • 13. Illy C, Quraishi O, Wang J, Purisima E, Vernet T, Mort J S. Role of the occluding loop in cathepsin B activity. J Biol Chem 1997; 272:1197-1202. PMID: 8995421, no PMCID, no DOI
  • 14. Choe Y, Leonetti F, Greenbaum D C, Lecaille F, Bogyo M, Bromme D, Ellman J A, Craik C S. Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities. J Biol Chem 2006; 281:12824-12832. PMID: 16520377, no PMCID, DOI: 10.1074/jbc.M513331200
  • 15. Gosalia D N, Salisbury C M, Maly D J, Ellman J A, Diamond S L. Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays. Proteomics 2005; 5:1292-1298. PMID: 15742319, no PMCID, DOI: 10.1002/pmic.200401011
  • 16. Harris J L, Backes B J, Leonetti F, Mahrus S, Ellman J A, Craik C S. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc Nat Acad Sci USA 2000; 97:7754-7759. PMID: 10869434, PMCID: PMC16617, DOI: 10.1073/pnas.140132697
  • 17. Khersonsky O, Tawfik D S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Ann Rev Biochem 2010; 79:471-505. PMID 20235827, no PMCID DOI: 10.1146/annurev-biochem-030409-143718
  • 18. Whitley M J, Cardona D M, Lazarides A L, Spasojevic I, Ferrer J M, Cahill J, Chang-Lung Lee C L, Matija Snuderl M, Dan G. Blazer D G III, E. Shelley Hwang E S, Greenup R A, Mosca P J, Mito J K, Cuneo K C, Larrier N A, O'Reilly E K, Riedel R F, Eward W C, Strasfeld D B, Fukumura D, Jain R K, Lee W D, Griffith L G, Bawendi M G, Kirsch D G, Brigman B E. A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer. Sci Trans Med 2016; 8:320ra4. PMID: 26738797, PMCID: PMC4794335, DOI: 10.1126/scitranslmed.aad0293
  • 19. Lazarides A L, Whitley M J, Strasfeld D B, Cardona D M, Ferrer J M, Mueller J L, Fu H L, DeWitt S B, Brigman B E, Ramanujam N, Kirsch D G, Eward W C. A fluorescence-guided laser ablation system for removal of residual cancer in a mouse model of soft tissue sarcoma. Theranostics 2016; 6:155-166. PMID: 26877775, PMCID: PMC4729765, DOI: 10.7150/thno.13536.
  • 20. Sheth R A, Upadhyay R, Stangenberg L, Sheth R, Weissleder R, Mahmood U. Improved detection of ovarian cancer metastases by intraoperative quantitative fluorescence protease imaging in a pre-clinical model. Gynecol Oncology 2009; 112:616-622. PMID: 19135233, PMCID: PMC2664404, DOI: 10.1016/j.ygyno.2008.11.018
  • 21. Nguyen Q T, Olson E S, Aguillera T A, Jiang T, Scadeng M, Ellies L G, Tsien R Y. Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival. Proc Nat Acad Sci USA. 2010; 107:4317-4322. PMID: 20160097, PMCID: PMC2840114, DOI: 10.1073/pnas.0910261107
  • 22. Mieog J S D, Hutteman M, van der Vorst J R, Kuppen P J K, Que I, Dijkstra J, Kaijzel E L, Prins F, Lowik CWGM, Smit VTHBM, van de Velde C J H, Vahrmeijer Al. Image-guided tumor resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer. Breast Cancer Res Treatment 2011; 128:679-689. PMID: 20821347, no PMCID, DOI: 10.1007/s10549-010-1130-6
  • 23. Mito J K, Ferrer J M, Brigman B E, Lee C L, Dodd R D, Eward W C, Marshall L F, Cuneo K C, Carter J E, Ramasunder S, Kim Y, Lee W D, Griffith L G, Bawendi M G, Kirsch D G. Interoperative detection and removal of microscopic residual sarcoma using wide-field imaging. Cancer 2012; 118:5320-5330. PMID: 22437667, PMCID: PMC3532657, DOI: 10.1002/cncr.27458
  • 24. Eward W C, Mito J K, Eward C A, Carter J E, Ferrer J M, Kirsch D G, Brigman B E. A novel imaging system permits real-time in vivo tumor bed assessment after resection of naturally occurring sarcomas in dogs. Clin Orthopaedics Related Res 2013; 471:834-842. PMID: 22972654, PMCID: PMC3563778, DOI: 10.1007/s11999-012-2560-8
  • 25. Blum G, von Degenfeld G, Merchant M J, Blau H M, Bogyo M. Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes. Nat Chem Biol 2007; 3:668-677. PMID: 17828252, no PMCID, DOI: 10.1038/nchembio.2007.26
  • 26. Blum G, Weimer R M, Edgington L E, Adams W, Bogyo M. Comparative assessment of substrates and activity based probes as tools for non-invasive optical imaging of cysteine protease activity. PloS One. 2009; 4:e6374. PMID: 19636372, PMCID: PMC2712068, DOI: 10.1371/journal.pone.0006374
  • 27. Olson E S, Jiang T, Aguilera T A, Nguyen Q T, Ellies L G, Scadeng M. Tsien R Y. Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc Natl Acad Sci USA 2010; 107:4311-6. PMID: 20160077, PMCID: PMC2840175, DOI: 10.1073/pnas.0910283107
  • 28. Fujii T, Kamiya M, Urano Y. In vivo imaging of intraperitoneally disseminated tumors in model mice by using activatable fluorescent small-molecular probes for activity of cathepsins. Bioconj Chem 2014; 25:1838-1846. PMID: 25196809, no PMCID, DOI: 10.1021/bc5003289
  • 29. Ofori L O, Withana N P, Prestwood T R, Verdoes M, Brady J J, Winslow M M, Sorger J, Bogyo M. Design of protease activated optical contrast agents that exploit a latent lysosomotropic effect for use in fluorescenceguided surgery. ACS Chem Biol 2015; 10:1977-1988. PMID: 26039341, PMCID: PMC4577961, DOI: 10.1021/acschembio.5b00205
  • 30. DeWitt S B, Eward W C, Eward C A, Lazarides A L, Whitley M J, Ferrer J M, Brigman B E, Kirsch D G, Berg J. A novel imaging system distinguishes neoplastic from normal tissue during resection of soft tissue sarcomas and mast cell tumors in dogs. Veterinary Surg 2016; 45:715-722. PMID: 27281113, PMCID: PMC4970890, DOI: 10.1111/vsu.12487
  • 31. Miampamba M, Liu J, Harootunian A, Gale A J, Baird S, Chen S L, Nguyen Q T, Tsien R Y, Gonzalez J E. Sensitive in vivo vlsualization of breast cancer using ratiometric protease-activatable fluorescent imaging agent, AVB-620. Theranostics 2017; 7:3369-3386. PMID: 28900516, PMCID: PMC5595138, DOI: 10.7150/thno.20678
  • 32. Ntziachristos V, Razansky D. Molecular imaging by means of multispectral optoacoustic tomography (MSOT). Chem Rev 2010; 110:2783-2794. PMID: 20387910, no PMCID, DOI: 10.1021/cr9002566
  • 33. Taruttis A, Ntziachristos V. Advances in real-time multispectral optoacoustic imaging and its applications. Nat Photon 2015; 9:219-227. No PMID, no PMCID, DOI: 10.1038/nphoton.2015.29
  • 34. Diot G, Metz S, Noske A, Liapis E, Schroeder B, Ovsepian S V, Meier R, Rummeny E J, Ntziachristos V. Multi-spectral optoacoustic tomography (MSOT) of human breast cancer. Clin Cancer Res 2017; 23:6912-6922. PMID: 28899968, no PMCID, DOI: 10.1158/1078-0432.CCR-16-3200.
  • 35. Becker A, MasthoffM, Claussen J, Ford S J, Roll W, Burg M, Barth P J, Heindel W, Schafers M, Eisenblatter M, Wildgruber M. Multispectral optoacoustic tomography of the human breast: characterization of healthy tissue and malignant lesions using a hybrid ultrasound-optoacoustic approach. Euro Radiol 2018; 28:602-609. PMID: 28786007, no PMCID, DOI: 10.1007/s00330-017-5002-x
  • 36. Neuschler E I, Butler R, Young C A, Barke L D, Bertrand M L, Bohm-Velez M, Destounis S, Donlan P, Grobmyer S R, Katzen J, Kist K A, Lavin P T, Makariou E V, Parris TM, Schilling K J, Tucker F L, Dogan B E. A pivotal study of optoacoustic imaging to diagnose benign and malignant breast masses: A new evaluation tool for radiologists. Radiology 2017; 287:398-412. PMID: 29178816, no PMCID, DOI: 10.1148/radio1.2017172228
  • 37. Waldner M J, Knieling F, Egger C, Morscher S, Claussen J, Vetter M, Kielisch C, Fischer S, Pfeifer L, Hagel A, Goertz R S, Wildner D, Atreya R, Strobel D, Neurath M F. Multispectral optoacoustic tomography in Crohn's disease: noninvasive imaging of disease activity. Gastroenterology 2016; 151:238-240. PMID: 27269244, no PMCID, DOI: 10.1053/j.gastro.2016.05.047
  • 38. Knieling F, Neufert C, Hartmann A, Claussen J, Urich A, Egger C, Vetter M, Fischer S, Pfeifer L, Hagel A, Kielisch C, Görtz R S, Wildner D, Engel M, Röther J, Uter W, Siebler J, Atreya R, Rascher W, Strobel D, Neurath M F, Waldner M J. Multispectral optoacoustic tomography for assessment of Crohn's diease activity. N Engl J Med 2017; 376:1294-1296. PMID: 28355498, no PMCID, DOI: 10.1056/NEJMc1612455
  • 39. He J, Yang L, Yi W, Fan W, Wen Y, Miao X, Xiong L. Combination of fluorescence-guided surgery with photodynamic therapy for the treatment of cancer. Mol Imaging 2017; 16:1536012117722911. PMID: 28849712, PMCID: PMC5580848, DOI: 10.1177/1536012117722911
  • 40. Shimizu K, Nitta M, Komori T, Maruyama T, Yasuda T, Fujii Y, Masamune K, Kawamata T, Maehara T, Muragaki Y. Intraoperative photodynamic diagnosis using talaporfin sodium simultaneously applied for photodynamic therapy against malignant glioma: a prospective clinical study. Front Neurol 2018; 9:24. PMID: 29441040, PMCID: PMC5797572, DOI: 10.3389/fneur.2018.00024
  • 41. Tsuda T, Kaibori M, Hishikawa H, Nakatake R, Okumura T, Ozeki E, Hara I, Morimoto Y, Yoshii K, Kon M. Near-infrared fluorescence imaging and photodynamic therapy with indocyanine green lactosome has antimeoplastic effects for hepatocellular carcinoma. Plos One 2017; 12:e0183527. PMID: 28859104, PMCID: PMC5578495, DOI: 10.1371/journal.pone.0183527
  • 42. Muhanna N, Cui L, Chan H, Burgess L, Jin C S, DacDonald T D, Huynh E, Wang F, Chen J, Irish J C, Zhang G. Multimodal image-guided surgical and photodynamic interventions in head and neck cancer: from primary tumor to metastatic drainage. Clin Cancer Res 2016; 22:961-970. PMID: 26463705, no PMCID, DOI: 10.1158/1078-0432.CCR-15-1235
  • 43. Mahmoudi K, Garvey K L, Bouras A, Cramer G, Stepp H, Raj J G J, Bozec D, Busch TM, Hadjipanayis C G. 5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas. J Neurooncol. 2019 February; 141(3):595-607. PMID: 30659522, PMCID: PMC6538286DOI: 10.1007/s11060-019-03103-4
  • 44. Zhou Z, Yan Y, Wang L, Zhang Q, Cheng Y. Melanin-like nanoparticles decorated with an autophagyinducing peptide for efficient targeted photothermal therapy. Biomaterials 2019; 203:63-72. PMID: 30852424, no PMCID, DOI: 10.1016/j.biomaterials.2019.02.023
  • 45. Fan B, Yang X, Li X, Lv S, Zhang H, Sun J, Li L, Wang L, Qu B, Peng X, Zhang R. Photoacoustic-imagingguided therapy of functionalized melanin nanoparticles: combination of photothermal ablation and gene therapy against laryngeal squamous cell carcinoma. Nanoscale 2019; 11:6285-6296. PMID: 30882835, no PMCID, DOI: 10.1039/c9nr01122f
  • 46. Kim M A, Yon S D, Kim E M, Jeong H J, Lee C M. Natural melanin-loaded nanovesicles for near-infrared mediated tumor ablation by photothermal conversion. Nanotech 2018; 29:415101. PMID: 30028309, no PMCID, DOI: 10.1088/1361-6528/aad4da
  • 47. Zhang L, Sheng D, Wang D, Yao Y, Yang K, Wang Z, Deng L, Chen Y. Bioinspired multifunctional melaninbased nanoliposome for photoacoustic/magnetic resonance imaging-guided efficient photothermal ablation of cancer. Theranostics 2018; 1591-1606. PMID: 29556343, PMCID: PMC5858169, DOI: 10.7150/thno.22430
  • 48. Cho S, Park W, Kim D H. Silica-coated metal chelating-melanin nanoparticles as a dual-modal contrast enhancement imaging and therapeutic agent. ACS Appl Mater Interfaces 2017; 9:101-111. PMID: 27992171, PMCID: PMC5509028, DOI: 10.1021/acsami.6b11304
  • 49. Shabat D. Self-immolative dendrimers as novel drug delivery platforms. J Poly Sci Part A 2006; 44:1569-1578. No PMID, no PMCID, DOI: 10.1002/pola.21258
  • 50. Blencowe C A, Russell A T, Greco F, Hayes W, Thornthwaite D W. Self-immolative linkers in polymeric delivery systems. Polymer Chem 2011; 2:773-790. No PMID, no PMCID, DOI: 10.1039/COPY00324G
  • 51. Rosen B M, Wilson C J, Wilson D A, Peterca M, Imam M R, Percec V. Dendron-mediated self-assembly, disassembly, and self-organization of complex systems. Chem Rev 2009; 109:6275-6540. PMID: 19877614, no PMCID, DOI: 10.1021/cr900157q
  • 52. Zheng H, Lin Z, Shi Y, Tian J, Wang F. Tyrosinase-based reporter gene for photoacoustic imaging of microRNA-9 regulated by DNA methylation in living subjects. Molec Therapy Nucl Acids 2018; 11:34-40. PMID: 29858069, PMCID: PMC5849859, DOI: 10.1016/j.omtn.2018.01.008
  • 53. Liu M, Wang Y, Li M, Feng H, Liu Q, Qin C, Zhang Y, Lan X. Using tyrosinase as a tri-modality reporter gene to monitor transplanted stem cells in acute myocardial infarction. Exp Molec Med 2018; 50:54. PMID: 29700279, PMCID: PMC5938053, DOI: 10.1038/s12276-018-0080-7
  • 54. Feng H, Xia X, Li C, Song Y, Qin C, Zhang Y, Lan X. TYR as a multifunctional reporter gene regulated by the Tet-on system for multimodality imaging: an in vitro study. Sci Rep 2015; 5:15502. PMID: 26483258, PMCID: PMC4611178, DOI: 10.1038/srep15502
  • 55. Paproski R J, Li Y, Barber Q, Lewis J D, Campbell R E, Zemp R. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli. J Biomed Optics 2015; 20:106008. PMID: 26502231, no PMCID, DOI: 10.1117/1.JBO.20.10.106008
  • 56. Paproski R J, Heinmiller A, Wachowicz K, Zemp R J. Multi-wavelength photoacoustic imaging of inducible tyrosinase reporter gene expression in xenograft tumors. Sci Rep 2014; 4:5329. PMID: 24936769; PMCID: PMC4060505, DOI: 10.1038/srep05329
  • 57. Qin C, Cheng K, Chen K, Hu X, Liu Y, Lan X, Zhang Y, Liu H, Xu Y, Bu L. Tyrosinase as a multifunctional reporter gene for photoacoustic/MRI/PET triple modality molecular imaging. Sci Rep 2013; 3:1490. PMID: 23508226, PMCID: PMC3603217, DOI: 10.1038/srep01490
  • 58. Krumholz A, VanVickle-Chavez S J, Yao J J, Fleming T P, Gillanders W E, Wang L H V. Photoacoustic microcopy of tyrosinase reporter gene in vivo. J biomed Optis 2011; 16:080503. PMID: 21895303, PMCID: PMC3162617, DOI: 10.1117/1.3606568
  • 59. Paproski R J, Forbrich A E, Wachowicz K, Hitt M M, Zemp R J. Tyrosinase as a dual reporter gene for both photoacoustic and magnetic resonance imaging. Biomed optics Express 2011; 2:771-780. PMID: 21483602, PMCID: PMC3072120, DOI: 10.1364/BOE.2.000771
  • 60. Levi J, Kothapalli S R, Ma T J, Hartman K, Khuri-Yakub B T, Gambhir S S. Design, synthesis and imaging of an activatable photoacoustic probe. J Am Chem Soc 2010; 132:11264-11269. PMID: 20698693, PMCID: PMC2922742, DOI: 10.1021/ja104000a
  • 61. Xia X, Yang M, Oetj en LK, Zhang Y, Li O, Chen J, Xia Y. An enzyme-sensitive probe for photoacoustic imaging and fluorescence detection of protease activity. Nanoscale 2011; 3:950-953. PMID: 21225037, no PMCID, DOI: 10.1039/c0nr00874e.
  • 62. Zha Z, Zhang S, Deng Z, Li Y, Li C, Dai Z. Enzyme-responsive copper sulphide nanoparticles for combined photoacoustic imaging, tumor-selective chemotherapy and photothermal therapy. Chem Comm 2013; 49:3455-457. PMID: 23507786, no PMCID, DOI: 10.1039/c3cc40608c
  • 63. Dragulescu-Andrasi A, Kothapalli S R, Tikhomirov G A, Rao J, Gambhir S S. Activatable oligomerizable imaging agents for photoacoustic imaging of furin-like activity in living subjects. J Am Chem Soc 2013; 135:11015-11022. PMID: 23859847, PMCID: PMC3771329, DOI: 10.1021/ja4010078
  • 64. Yang K, Zhu L, Nie L, Sun X, Cheng L, Wu C, Niu G, Chen X, Liu Z. Visualization of protease activity in vivo using an activatable photoacoustic imaging probe based on CuS nanoparticles. Theranostics 2014; 4:134-141. PMID: 24465271, PMCID: PMC3900798, DOI: 10.7150/thno.7217
  • 65. Hirasawa T, Iwatate R J, Kamiya M, Okawa S, Urano Y, Ishihara M. Multispectral photoacouostic imaging of tumours in mice injected with an enzyme-activatable photoacoustic probe. J Optics 2017; 19:014002. PMID: 29736563, no PMCID, DOI: 10.1007/s11307-018-1203-1
  • 66. Piletic I R, Matthews T E, Warren W S. Estimation of molar absorptivities and pigment sizes for eumelanin and pheomelanin using femtosecond transient absorption spectroscopy. J Chem Phys 2009; 131:181106. PMID: 19916591, PMCID: PMC4108625, DOI: 10.1063/1.3265861
  • 67. Solano F. Melanin and melanin-related polymers as materials with biomedical and biotechnological applications—cuttlefish ink and mussel foot proteins as inspired biomolecules. Int J Molec Sciences 2017; 18:1561. PMID: 28718807, PMCID: PMC5536049, DOI: 10.3390/ijms18071561
  • 68. Zhang R, Fan Q, Yang M, Cheng K, Lu X, Zhang L, Huang W, Cheng Z. Engineering melanin nanoparticles as an efficient drug-delivery system for imaging-guided chemotherapy. 2015; 27:5063-5069. PMID: 26222210, no PMCID, DOI: 10.1002/adma.201502201
  • 69. Liopo A, Su R, Orafesky A A, Melanin nanoparticles as a novel contrast agent for optoacoustic tomography. Photoacoustics 2015; 3:35-43. No PMID, no PMCID, DOI: 10.1016/j.pacs.2015.02.001
  • 70. Fan Q, Cheng K, Hu X, Ma X, Zhang R, Yang M, Lu X, Xing L, Huuang W, Gambhir S S, Cheng Z. Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging. J Am Chem Soc 2014; 136:15185-15194. PMID: 25292385, PMCID: PMC4227813, DOI: 10.1021/ja505412p
  • 71. Longo D L, Stefania R, Aime S, Oraevsky A. Melanin-based contrast agents for biomedical optoacoustic imaging and theranostic applications. Int J Molec Sci 2017; 18:1719. PMID: 28783106, PMCID: PMC5578109, DOI: 10.3390/ijms18081719
  • 72. Li Y, Sheth V R, Liu G, Pagel M D. A self-calibrating PARACEST MM contrast agent that detects esterase enzyme activity. Contrast Media Molec Imaging 2011; 6:219-228. PMID 21861282, PMCID: PMC4879975, DOI: 10.1002/cmmi.421
  • 73. Fernandez-Cuervo G, Tucker K A, Malm S W, Jones K M, Pagel M D.

Diamagnetic imaging agents with a modular chemical design for quantitative detection of β-galactosidase and β-glucuronidase activities with catalyCEST MM. Bioconj Chem 2016, 27:2549-2557. PMID: 27657647, no PMCID, DOI: 10.1021/acs.bioconjchem.6b00482.

  • 74. Cox B, Laufer J G, Arridge S R, Beard P C. Quantitative spectroscopic photoacoustic imaging: a review. J Biomed Opt 2002; 17:061202. PMID: 22734732, no PMCID, DOI: 10.1117/1.JBO.17.6.061202
  • 75. Lutzweiler C, Razansky D. Optoacoustic imaging and tomography: reconstruction approaches and outstanding challenges in image performance and quantification. Sensors 2013; 13:7345-7384. PMID: 23736854, PMCID: PMC3715274, DOI: 10.3390/s130607345
  • 76. Brochu F M, Brunker J, Joseph J, Tomaszewski M, Morscher S, Bohndiek S E. Towards quantitative evaluation of tissue absorption coefficients using light fluence correction in optoacoustic tomography. IEEE Trans Med Imaging 2017; 36:322-331. PMID: 27623576, no PMCID, DOI: 10.1109/TMI.2016.2607199
  • 77. Hupple C W, Morscher S, Burton N C, Pagel M D, McNally L R, Cardenas-Rodriguez J. A light-fluenceindependent method for the quantitative analysis of dynamic contrast-enhanced multispectral optoacoustic tomography (DCE MSOT). Photoacoustics 2018; 10:54-64. No PMID, no PMCID, DOI: 10.1016/j.pacs.2018.04.003
  • 78. Sheth R A, Upadhyay R, Stangenberg L, Sheth R, Weissleder R, Mahmood U. Improved detection of ovarian cancer metastases by intraoperative quantitative fluorescence protease imaging in a pre-clinical model. Gynecol Oncol 2009; 112:616-22. PMID: 19135233, PMCID: PMC2664404, DOI: 10.1016/j.ygyno.2008.11.018
  • 79. Nguyen Q T, Olson E S, Aguilera T A, Jiang T, Scadeng M, Ellies L G, Tsien R Y. Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival. Proc Nat Acad Sci USA 2010; 107:4317-4322. PMID: 20160097, PMCID: PMC2840114, DOI: 10.1073/pnas.0910261107
  • 80. Mieog J S, Hutteman M, van der Vorst J R, Kuppen P J, Que I, Dijkstra J, Kaijzel E L, Prins F, Lowik C W, Smit V T, van de Velde C J, Vahrmeijer A L. Image-guided tumor resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer. Breast Cancer Res Treat 2011; 128:679-689. PMID: 20821347, on PMCID, DOI: 10.1007/s10549-010-1130-6.
  • 81. Mito J K, Ferrer J M, Brigman B E, Lee C L, Dodd R D, Eward W C, Marshall L F, Cuneo K C, Carter J E, Ramasunder S, Kim Y, Lee W D, Griffith L G, Bawendi M G, Kirsch D G. Intraoperative detection and removal of microscopic residual sarcoma using wide-field imaging. Cancer 2012; 118; 5320-5330. PMID: 22437667, PMCID: PMC3532657, DOI: 10.1002/cncr.27458
  • 82. Savariar E N, Felsen C N, Nashi N, Jiang T, Ellies L G, Steinbach P, Tsien R Y, Nguyen Q. Real-time in vivo molecular detection of primary tumors and metastases with ratiometric activatable cell-penetrating peptides. Cancer Res 2013; 73:855-64. PMID: 23188503; PMCID: PMC3799878. DOI: 10.1158/0008-5472.CAN-12-2969
  • 83. Hu H Y, Vats D, Vizovisek M, Kramer L, Germanier C, Wendt K U, Rudin M, Turk B, Plettenburg O, Schultz C. In vivo imaging of mouse tumors by a lipidated cathepsin S substrate. Angew Chem Int Ed 2014; 53:7669-7673. PMID: 24888522, PMCID: PMC4298799, DOI: 10.1002/anie.201310979
  • 84. Fujii T, Kamiya M, Urano Y. In vivo imaging of intraperitoneally disseminated tumors in model mice by using activatable fluorescent small-molecular probes for activity of cathepsins. Bioconj Chem 2014; 25:1838-1846. PMID: 25196809, no PMCID, DOI: 10.1021/bc5003289 85. Ofori L O, Withana N P, Prestwood T R, Verdoes M, Brady J J, Winslow M M, Sorger J, Bogyo M. Design of protease activated optical contrast agents that exploit a latent lysosomotropic effect for use in fluorescenceguided surgery. ACS Chem Biol 2015; 10:1977-1988. PMID: 26039341, PMCID: PMC4577961, DOI: 10.1021/acschembio.5b00205
  • 86. Segal E, Prestwood T R, van der Linden W A, Carmi Y, Bhattacharya N, Withana N, Verdoes M, Habtezion A, Engleman E G, Bogyo M. Detection of intestinal cancer by local, topical application of a quenched fluorescence probe for cysteine cathepsins. Chem Biol 2015; 22:148-158. PMID: 25579207, PMCID: PMC4353655, DOI: 10.1016/j.chembio1.2014.11.008
  • 87. Chi C, Zhang Q, Mao Y, Kou D, Qiu J, Ye J, Wang J, Wang Z, Du Y, Tian J. Increased precision of orthotopic and metastatic breast cancer surgery guided by matrix metalloproteinase-activatable nearinfrared fluorescence probes. Sci Rep 2015; 5:14197. PMID: 26395067, PMCID: PMC4585795, DOI: 10.1038/srep14197
  • 88. Metildi C A, Felsen C N, Savariar E N, Nguyen Q, Kaushal S, Hoffman R M, Tsien R Y, Bouvet M. Ratiometric activatable cell-penetrating peptides label pancreatic cancer, enabling fluorescence-guided surgery, which reduces metastases and recurrence in orthotopic mouse models. Ann Surg Oncol 2015; 22:2082-2087. PMID: 25319581; PMCID: PMC4400250, DOI: 10.1245/s10434-014-4144-1
  • 89. Whitley M J, Cardona D M, Lazarides A L, Spasojevic I, Ferrer J M, Cahill J, Lee C L, Snuderi M, Blazer D G, Hwang E S, Greenup R A, Mosca P J, Mito J K, Cuneo K C, Lamer N A, O'Reilly E K, Riedel R F, Eward W C, Strasfeld D B, Fukumura D, Jain R K, Lee W D, Griffith L G, Bawendi M G, Kirsch D G, Brigman B E. A mousehuman phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer. Sci Transl Med 2016; 8:320ra324. PMID: 26738797, PMCID: PMC4794335, DOI: 10.1126/scitranslmed.aad0293
  • 90. Orosco R K, Savariar E N, Weissbrod P A, Diaz-Perez J A, Bouvet M, Tsien R Y, Nguyen Q. Molecular targeting of papillary thyroid carcinoma with fluorescently labeled ratiometric activatable cell penetrating peptides in a transgenic murine model. J Surg Oncol 2016; 113:138-143. PMID: 26799257; PMCID: PMC4986916, DOI: 10.1002/jso.24129
  • 91. Hussain T, Savariar E N, Diaz-Perez J A, Messer K, Pu M, Tsien R Y, Nguyen Q. Surgical molecular navigation with ratiometric activatable cell penetrating peptide for intraoperative identification and resection of small salivary gland cancers. Head Neck 2016; 38:715-723. PMID: 25521629; PMCID: PMC4472578, DOI: 10.1002/hed.23946
  • 92. Unkart J T, Chen S L, Wapnir I L, Gonzalez J E, Harootunian A, Wallace A M. Intraoperative tumor detection using a ratiometric activatable fluorescent peptide: a first-in-human phase 1 study. Ann Surg Oncol 2017; 24:3167-73. PMID: 28699134, no PMCID, DOI: 10.1245/s10434-017-5991-3
  • 93. Miampamba M, Liu J, Harootunian A, Gale A J, Baird S, Chen S L, Nguyen Q, Tsien R Y, Gonzalez J E. Sensitive in vivo visualization of breast cancer using ratiometric protease-activatable fluorescent imaging agent, AVB-620. Theranostics 2017; 7:3369-3386. PMID: 28900516, PMCID: PMC5595138, DOI: 10.7150/thno.20678
  • 94. Hingorani D V, Lemieux A J, Acevedo J R, Glasgow H L, Kedarisetty S, Whitney M A, Molinolo A A, Tsien R Y, Nguyen Q. Early detection of squamous cell carcinoma in carcinogen induced oral cancer rodent model by ratiometric activatable cell penetrating peptides. Oral Oncol 2017; 71:156-162. PMID: 28688684, PMCID: PMC5575898, DOI: 10.1016/j.oraloncology.2017.06.009
  • 95. Reaction A: Boger D L, Yohannes D. Selectively protected L-DOPA derivatives: Application of benzylic hydroperoxide rearrangement. J Org Chem 1987; 52:5283-5286. No PMID, no PMCID, no DOI.
  • 96. Reaction B: (a) Wuts P G M, Greene T W. Greene's Protective Groups in Organic Synthesis, 4th Edition. John Wiley & Sons, Inc., New York, N.Y., 2006. No PMID, no PMCID, no DOI. (b) He G, Lu C, Zhao Y, Nack W A, Chen G. Improved protocol for indoline synthesis via palladiumcatalyzed intramolecular C(sp2)-H amination. Org Lett 2012; 14:2944-2947. PMID: 22670815, no PMCID, DOI: 10.1021/01301352v
  • 97. Reaction C: (a) Kendall P M, Johnson J V, Cook C E. Synthetic route to an aromatic analog of strigol. J Org Chem 1979; 44:1421-2424. No PMID, no PMCID, no DOI. (b) Ronald R C, Lasinger J M, Lillie T S, Wheeler C J. Total synthesis of frustulosin and aurocitrin. J Org Chem 1082; 47; 2541-2549. No PMID, no PMCID, no DOI.
  • 98. Reaction D: He G, Lu C, Zhao Y, Nack W A, Chen G. Improved protocol for indoline synthesis via palladium-catalyzed intramolecular C(sp2)-H amination. Org Lett 2012; 14:2944-2947. PMID: 22670815, no PMCID, DOI: 10.1021/01301352v
  • 99. Reaction E: Chen Y F, Wilcoxen K. An improved synthesis of selectively protected L-DOPA derivatives from L-tyrosine. J Org Chem 2000; 65:2574-2576. PMID: 10789475, no PMCID, no DOI.

100. Reaction F: Vezenkov L, Honson N S, Kumar N S, Bosc D, Kovacic S, nguyen TG, Pfeifer T A, Young R N. Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B. Bioorg Med Chem 2015; 23:3237-3247. PMID: 25979376, no PMCID, DOI: 10.1016/j.bmc.2015.04.064

  • 101. Reaction G: Eichberg M J, Dorta R L, Lamottke K, Vollhardt K P C. The formal total synthesis of (+−)-strychnine via a cobalt-mediated [2+2+2]cycloaddition. Org Lett 2000; 2:2479-2481. PMID: 10956526, no PMCID, no DOI.
  • 102. Reaction H: Ito S, Inoue S, Yamamoto Y, Fujita K. Synthesis and antitumor activity of cysteinyl-3,4-dihydroxyphenylalanines and related compounds. J Med Chem 1981; 24:673-677. PMID: 6788955, no PMCID, no DOI
  • 103. Reaction I: Wuts P G M, Greene T W. Greene's Protective Groups in Organic Synthesis, 4th Edition. John Wiley & Sons, Inc., New York, N.Y., 2006. No PMID, no PMCID, no DOI.
  • 104. Pagel M D. Structures and related properties of helical, disulfide-stabilized peptides. Ph.D. dissertation. Lawrence Berkeley Laboratory, University of California, Berkeley, 1993. Structures and related properties of helical, disulfide-stabilized peptides.
  • 105. Vezenkov L, Honson N S, Kumar N S, Bosc D, Kovacic S, nguyen TG, Pfeifer T A, Young R N. Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B. Bioorg Med Chem 2015; 23:3237-3247. PMID: 25979376, no PMCID, DOI: 10.1016/j.bmc.2015.04.064
  • 106. Yoo B, Pagel M D. Peptidyl molecular imaging contrast agents using a new solid phase peptide synthesis approach. Bioconj Chem 2007; 18:903-911. PMID: 17330953, PMCID: PMC2584118, DOI: 10.1021/bc060250q
  • 107. Yoo B, Pagel M D. A facile synthesis of α-amino-DOTA as a versatile molecular imaging probe. Tet Lett 2006; 47:7327-7330. No PMID, no PMCID, DOI: 10.1016/j.tetlet.2006.08.019
  • 108. Liu G, Li Y, Pagel M D. Design and characterization of new irreversible responsive PARACEST Mill contrast agent that detects nitric oxide. Magn Reson Med 2007; 58:1249-1256. PMID 18046705, no PMCID, DOI: 10.1002/mrm.21428
  • 109. Yoo B, Sheth V, Pagel M D. An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis. Tet Lett 2009; 50:4459-4462. PMID 20161272, PMCID: PMC2702766, DOI: 10.1016/j.tetlet.2009.05.061
  • 110. Ali M M, Yoo B, Pagel M D. Tracking the relative in vivo pharmacokinetics of nanoparticles with PARACEST MM. Molec Pharmaceutics 2009; 6:1409-1416. PMID 19298054, PMCID: PMC4216567, DOI: 10.1021/mp900040u
  • 111. Hingorani D V, Randtke E A, Pagel M D. A catalyCEST MM contrast agent that detects the enzymecatalyzed creation of a covalent bond. J Am Chem Soc 2013; 135:6396-6398. PMID 23601132, PMCID: PMC3667154, DOI: 10.1021/j a400254e.
  • 112. Yoo B, Sheth V R, Howison C M, Douglas M J K, Pineda C T, Maine E A, Baker A F, Pagel M D. Detection of in vivo enzyme activity with catalyCEST MM. Mag Reson Med 2014; 71:1221-1230. PMID 23640714, PMCID: PMC3742626, DOI: 10.1002/mrm.24763.
  • 113. Fernandez-Cuervo G, Sinharay S, Pagel M D. A catalyCEST Mill contrast agent that can simultaneously detect two enzyme activities. ChemBioChem, 2016; 17(5):383-387. PMID: 26693680, PMCID: PMC4814164, DOI: 10.1002/cbic.201500586.
  • 114. Sinharay S, Fernandez-Cuervo G, Acfalle J P, Pagel M D. Detection of sulfatase enzyme activity with a catalyCEST MM contrast agent. Chem Euro J 2016; 22:6491-6495. PMID: 26956002, PMCID: PMC4877021, DOI: 10.1002/chem.201600685
  • 115. Daryaei I, Randtke E A, Pagel M D. A biomarker-responsive T2ex MM contrast agent. Magn Reson Med 2017; 77:1665-1670. PMID: 27090199, PMCID: PMC5071101, DOI: 10.1002/mrm.26250
  • 116. Sinharay S, Randtke E A, Jones K M, Howison C M, Chambers S K, Kobayashi H, Pagel M D. Noninvasive detection of enzyme activity in tumor models of human ovarian cancer using catalyCEST MM. Magn Reson Med 2017; 77:2005-2014. PMID: 27221386, PMCID: PMC5123981, DOI: 10.1002/mrm.26278
  • 117. Daryaei I, Jones K M, Pagel M D. Detection of DT-diaphorase enzyme with a paraCEST MRI contrast agent. Chem Euro J 2017; 23:6514-6517. PMID: 28370655, no PMCID, DOI: 10.1002/chem.201700721.
  • 118. Sinharay S, Howison C M, Baker A F, Pagel M D. Detecting in vivo urokinase Plasminogen Activator activity with a catalyCEST MM contrast agent. NMR Biomed 2017, 30(7):e3721. PMID: 28370884, PMCID: PMC5704996, DOI: 10.1002/nbm.3721
  • 119. Sinharay S, Randtke E A, Howison C M, Ignatenko N A, Pagel M D. Detection of enzyme activity and inhibition during studies in solution, in vitro and in vivo with catalyCEST MM. Molec Imaging Biol 2018; 20:240-248. PMID: 28726131, no PMCID, DOI: 10.1007/s11307-017-1092-8
  • 120. Blencowe C A, Russell A T, Greco F, Hayes W, Thornwaite D W. Self-immolative linkers in polymeric delivery systems. Polymer Chem 2011; 2:773-790. No PMID, no PMCID, DOI: 10.1039/COPY00324G 121. Yoo B, Pagel M D. A PARACEST MRI contrast agent to detect enzyme activity. J Am Chem Soc 2006; 128:14032-14033. PMID 17061878, no PMCID, DOI: 10.1021/ja063874f
  • 122. Yoo B, Raam M, Rosenblum R, Pagel M D. Enzyme-responsive PARACEST MRI contrast agents: A new biomedical imaging approach for studies of the proteasome. Contrast Media Molec Imaging 2007; 2:189-198. PMID 17712869, no PMCID, DOI: 10.1002/cmmi.145
  • 123. Cat B Fast commercial product information: http://www.perkinelmer.com/lab-products-andservices/application-support-knowledgebase/in-vivo-preclinical-imaging/cat-b-fast-ask.html
  • 124. Sensolyte—AFC® Urokinase (uPA) Activity Assay Kit commercial product information: https://www.anaspec.com/products/product.asp?id=51781
  • 125. Withana N P, Blum G, Sameni M, Sameni M, Slaney C, Anbalagan A, Olive M B, Bidwell B N, Edgington L, Wang L, Moin K, Sloane B F, Anderson R L, Bogyo M S, Parker B S. Cathepsin B inhibition limits bone metastasis in breast cancer. Cancer Res 2012; 72:1199-1209. PMID: 22266111, PMCID: PMC3538126, DOI: 10.1158/0008-5472.CAN-11-2759
  • 126. Billstrom A, Hartley-Asp B, Lecander I, Batra S, Astedt B. The urokinase inhibitor p-aminobenzamidine inhibits growth of a human prostate tumor in SCID mice. Int J Cancer 1995; 61:542-547. PMID: 7759160, no PMCID, no DOI
  • 127. Randtke E A, Chen L Q, Corrales L R, Pagel M D. The Hanes-Woolf Linear QUESP method improves the measurements of fast chemical exchange rates with CEST MRL Magn Reson Med 2014; 71:1603-1612. PMID 23780911, PMCID: PMC3784632, DOI: 10.1002/mrm.24792.
  • 128. Xing R, Addington A K, Mason R W. Quantification of cathepsins B and L in cells. Biochem J 1998; 332:499-505. PMID: 9601080, PMCID: PMC1219506, DOI: 10.1042/bj3320499
  • 129. Zajc I, Frangež L, Lah T T. Expression of cathepsin B is related to tumorigenicity of breast cancer cell lines. Radiol Oncol 2003; 37:233-240. PMID: 12715895, no PMCID, DOI: 10.1515/BC.2003.050
  • 130. Bajou K, Lewalle J M, Martinez C R, Soria C, Lu H, Noel A, Foidart J M. Human breast adenocarcinoma cell lines promote angiogenesis by providing cells with uPA-PAI-1 and by enhancing their expression. Int J Cancer 2002; 100:501-506. PMID: 12124797, no PMCID, DOI: 10.1002/ijc.10487
  • 131. LeBeau A M, Sevillano N, King M L, Duriseti S, Murphy S T, Craik C S, Murphy L L, VanBrocklin HF. Imaging the urokinase plasminongen activator receptor in preclinical breast cancer models of acquired drug resistance. Theranostics 2014; 4:267-279. PMID: 24505235, PMCID: PMC3915090, DOI: 10.7150/thno.7323
  • 132. Sliutz G, Eder H, Koelbl H, Tempfer C, Auerbach L, Schneeberger C, Kainz C, Zeillinger R. Quantification of uPA receptor expression in human breast cancer cell lines by cRT-PCR. Breast Cancer Res Treatment 1996; 40:257-263. PMID: 8883968, no PMCID, no DOI.
  • 133. Huber M C, Mall R, Braselmann H, Feuchtinger A, Molatore S, Lindner K, Walch A, Gross E, Schmitt M, Falkenberg N, Aubele M. uPAR enhances malignant potential of triple-negative breast cancer by directly interacting with uPA and IGF1R. BMC Cancer 2016; 16:615. PMID: 27502396, PMCID: PMC4977758, DOI: 10.1186/s12885-016-2663-9
  • 134. Bishop J A, Nelson A M, Merz W G, Askin F B, Riedel S. Evaluation of the detection of melanin by the Fontana-Masson silver stain in tissue with a wide range of organisms including Cryptococcus. Hum Pathol 2012; 43:898-903. PMID: 22154051, no PMCID, DOI: 10.1016/j.humpath.2011.07.021
  • 135. Kobes J E, Daryaei I, Howison C M, Bontrager J G, Siriani R S, Meuillet E M, Pagel M D. Improved treatment of pancreatic cancer with drug delivery nanoparticles loaded with a novel AKT/PDK1 inhibitor. Pancreas 2016; 45:1158-1166. PMID: 26918875, PMCID: PMC4983222, DOI: 10.1097/MPA.0000000000000607.
  • 136. Miller L R, Marks C, Becker J B, Hum P D, Chen W J, Woodruff T, McCarthy M M, Sohrabji F, Schiebinger L, Wetherington C L, Makris S, Arnold A P, Einstein G, Miller V M, Sandberg K, Maier S, Cornelison T L, Clayton J A. Considering sex as a biological variable in preclinical research. FASEB J, 2017; 31:29-34. PMID: 27682203, no PMCID, DOI: 10.1096/fj.201600781R
  • 137. Alford R, Simpson H M, Dubeman J, Hill G C, Ogawa M, Regino C, Kobayashi H, Choyke P L. Toxicity of organic fluorophores used in molecular imaging: Literature review. Mol Imaging 2009; 8:341-354. PMID: 20003892, no PMCID, no DOI.
  • 138. Chen L Q, Randtke E A, Jones K M, Moon B F, Howison C M, Pagel M D. Evaluations of tumor acidosis within in vivo tumor models using parametric maps generated with acidoCEST MRI. Mol Imaging Biol 2015; 17:488-496. PMID: 25622809, PMCID: PMC4880367, DOI: 10.1007/s11307-014-0816-2.

Detection of Hydrolase Activity

  • 1. Gambhir S S, Yaghoubi S S. Molecular Imaging with Reporter Genes. Cambridge University Press, 2010. No PMID, no PMCID, DOI: 10.1017/CB09780511730405
  • 2. Bourdeau R W, Lee-Gosselin A, Lakshmanan A, Farhadi A, Kumar S R, Nety S P, Shapiro M G. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature 2018; 553:86-90. PMID: 29300010, PMCID: PMC5920530, DOI: 10.1038/nature25021
  • 3. Ntziachristos V, Razansky D. Molecular imaging by means of multispectral optoacoustic tomography (MSOT). Chem Rev 2010; 110:2783-2794. PMID: 20387910, no PMCID, DOI: 10.1021/cr9002566
  • 4. Taruttis A, Ntziachristos V. Advances in real-time multispectral optoacoustic imaging and its applications. Nat Photon 2015; 9:219-227. No PMID, no PMCID, DOI: 10.1038/nphoton.2015.29
  • 5. Mason H S. A classification of melanins. Ann New York Acad Sci 1948; 4:399-404. PMID: 18862181, no PMCID, no DOI.
  • 6. del Marmol V, Beermann F. Tyrosinase and related proteins in mammalian pigmentation. FEBS Lett 1996; 382:165-168. PMID: 8601447, no PMCID, no DOI.
  • 7. Cox B, Laufer J G, Arridge S R, Beard P C. Quantitative spectroscopic photoacoustic imaging: a review. J Biomed Opt 2002; 17:061202. PMID: 22734732, no PMCID, DOI: 10.1117/1.JBO.17.6.061202
  • 8. Lutzweiler C, Razansky D. Optoacoustic imaging and tomography: reconstruction approaches and outstanding challenges in image performance and quantification. Sensors 2013; 13:7345-7384. PMID: 23736854, PMCID: PMC3715274, DOI: 10.3390/s130607345
  • 9. Brochu F M, Brunker J, Joseph J, Tomaszewski M, Morscher S, Bohndiek S E. Towards quantitative evaluation of tissue absorption coefficients using light fluence correction in optoacoustic tomography. IEEE Trans Med Imaging 2017; 36:322-331. PMID: 27623576, no PMCID, DOI: 10.1109/TMI.2016.2607199
  • 10. Hall C V, Jacob P E, Ringold G M, Lee F. Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Molec App Gen 1983; 2:101-109. PMID: 6302193, no PMCID, no DOI.
  • 11. Jefferson R A. Burgess S M. Hirsh D. Beta-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Nat Acad Sci USA 1986; 83: 8447-8451. PMID 3534890, PMCID: PMC386947, DOI: 10.1073/pnas.83.22.8447.
  • 12. Eustice D C, Feldman P A, Colberg-Poley A M, Buckery R M, Neubauer R H. A sensitive method for the detection of beta-galactosidase in transfected mammalian cells. Biotechniques 1991; 11:739-740. PMID: 1809326, no PMCID, no DOI.
  • 13. Louie A Y, Huber M M, Ahrens E T, Rothbacher U, Moats R, Jacobs R E, Fraser S E, Meade T E. In vivo visualization of gene expression using magnetic resonance imaging. Nat Biotechnol 2000; 18:321-325. PMID: 10700150, no PMCID, DOI: 10.1038/73780
  • 14. Tung, C. H., Zeng, Q., Shah, K., Kim, D E, Schellingerhout D, Weissleder R. In vivo imaging of beta-galactosidase activity using far red fluorescent switch. Cancer Res 2004; 64:1579-1583. PMID: 14996712, no PMCID, no DOI
  • 15. Wehrman T S, von Degenfeld G, Krutzik P O, Nolan G P, Blau H M. Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence. Nat Methods. 2006; 3:295-301. PMID: 16554835, no PMCID, DOI: 10.1038/nmeth868
  • 16. Kodibagkar V D, Yu J, Liu L, Hetherington H P, Mason R P. Imaging beta-galactosidase activity using 19F chemical shift imaging of LacZ gene-reporter molecule 2-fluoro-4-nitrophenol-beta-D-galactopyranoside. Magn Reson Imaging. 2006; 24:959-962. PMID: 16916713, no PMCID, DOI: 10.1016/j.mri.2006.04.003
  • 17. Li L, Zemp R J, Lungu G, Stoica G, Wang L V. Photoacoustic imaging of lacZ gene expression in vivo. J Biomed Opt 2007; 12:020504. PMID: 17477703, no PMCID, DOI: 10.1117/1.2717531
  • 18. Su Y C, Chuang K H, Wang Y M, Cheng C M, Lin S R, Wang J Y, Hwang J J, Chen B M, Chen K C, Roffler S, Cheng T L. Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane anchored 3-glucuronidase. Gene Ther 2007; 14:565-574. PMID: 17235292, no PMCID, DOI: 10.1038/sj.gt.3302896
  • 19. van Dort M E, Lee K C, Hamilton C A, Rehemtulla A, Ross B D. Radiosynthesis and evaluation of 5-[125I]iodoindol-3-yl-beta-D-galactopyranoside as a beta-galactosidase imaging radioligand. Mol Imaging. 2008; 7:187-197. PMID: 19123989, PMCID: PMC2743942, no DOI.
  • 20. Celen S, Cleynhens J, Deroose C, de Groot T, Ibrahimi A, Gijsbers R, Debyser Z, Mortelmans L, Verbruggen A, Bormans G. Synthesis and biological evaluation of (11)C-labeled beta-galactosyl triazoles as potential PET tracers for in vivo LacZ reporter gene imaging. Bioorg Med Chem. 2009; 17:5117-5125. PMID: 19515568, no PMCID, DOI: 10.1016/j.bmc.2009.05.056
  • 21. Zhang G J, Chen T B, Connolly B, Lin S A, Hargreaves R, Vanko A, Bednar B, Macneil D J, Sur C, Williams D L. In vivo optical imaging of LacZ expression using lacZ transgenic mice. Assay Drug Dev Technol. 2009; 7:391-399. PMID: 19689207, no PMCID, DOI: 10.1089/adt.2009.0195
  • 22. Tzou S C, Roffler S, Chuang K H, Yeh H P, Kao C H, Su Y C, Cheng C M, Tseng W L, Shiea J, Harm I H, Cheng K W, Chen B M, Hwang J J, Cheng T L, Wang H E. Micro-PET imaging of β-glucuronidase activity by the hydrophobic conversion of a glucuronide probe. Radiology 2009; 252:754-62. PMID: 19717754, no PMCID, DOI: 10.1148/radiol.2523082055
  • 23. Liu L, Mason R P. Imaging beta-galactosidase activity in human tumor xenografts and transgenic mice using a chemiluminescent substrate. PLoS One 2010; 5:e12024. DOI: 10.1371/journal.pone.0012024. PMID: 20700459, PMCID: PMC2917367, DOI: 10.1371/journal.pone.0012024
  • 24. Cui W, Liu L, Kodibagkar V D, Mason R P. Magn Reson Med 2010. S-Gal®, a novel 1H Mill reporter for β-galactosidase. Magn Reson Med 2010; 64:65-71. DOI: 10.1002/mrm.22400. PMID: 20572145, PMCID: PMC2924164, DOI: 10.1002/mrm.22400
  • 25. Antunes I F, Haisma H J, Elsinga P H, van Waarde A, Willemsen A T, Dierckx R A, de Vries E F. In vivo evaluation of 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-D-glucopyronuronate: a positron emission tomographic tracer for imaging β-glucuronidase activity in a tumor/inflammation rodent model. Mol. Imaging 2012; 11:77-87. PMID: 22418030, no PMCID, no DOI.
  • 26. Su Y C, Cheng T C, Leu Y L, Roffler S R, Wang J Y, Chuang C H, Kao C, Chen K C, Wang H E, Cheng T L. PET imaging of β-glucuronidase activity by an activity-based 124I-trapping probe for the personalized glucuronide prodrug targeted therapy. Mol Cancer Ther 2014; 13:2852-2863. PMID: 25277385, no PMCID, DOI: 10.1158/1535-7163.MCT-14-0212
  • 27. Asanuma D, Sakabe M, Kamiya M, Yamamoto K, Hiratake J, Ogawa M, et al. Sensitive beta-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo. Nat Commun (2015) 6:6463. PMID: 25765713, PMCID: PMC4382686, DOI: 10.1038/ncomms7463
  • 28. Fernández-Cuervo G, Tucker K A, Maim S W, Jones K M, Pagel M D. Diamagnetic imaging agents with a modular chemical design for quantitative detection of β-galactosidase and β-glucuronidase activities with catalyCEST MM. Bioconj Chem 2016, 27:2549-2557. PMID: 27657647, no PMCID, DOI: 10.1021/acs.bioconjchem.6b00482.
  • 29. Valluru K S, Wilson K E, Willman J K. Photoacoustic imaging in oncology: Translational preclinical and early clinical experience. Radiology 2016; 280:332-349. PMID: 27429141, PMCID: PMC4976462, DOI: 10.1148/radio1.16151414
  • 30. Buehler A, Dean-ben XL, Claussen J, Ntziachristos V, Razansky D. Three-dimensional optoacoustic tomography at video rate. Opt Express 2012; 20:22712-22719. PMID: 23037421, no PMCID, no DOI.
  • 31. Taruttis A, Morscher S, Burton N C, Razansky D, Ntziachristos V. Fast multispectral optoacoustic tomography (MSOT) for dynamic imaging of pharmacokinetics and biodistribution in multiple organs. PLoS ONE 2012; 7:e30491. PMID: 22295087, PMCID: PMC3266258, DOI: 10.1371/journal.pone.0030491
  • 32. Dean-Ben X L, Bay E, Razansky D. Functional optoacoustic imaging of moving objects using microsecond-delay acquisition of multi spectral three-dimensional tomographic data. Sci Rep 2014; 4:5878. PMID: 25073504, PMCID: PMC4115207, DOI: 10.1038/srep05878
  • 33. Fehm T E, Dean-Ben X L, Ford S J, Razansky D. In vivo whole-body optoacoustic scanner with real-time volumetric imaging capacity. Optica 2016; 3:1153-1159. No PMID, no PMCID, DOI: 10.1364/OPTICA.3.001153
  • 34. Krumholz A, VanVickle-Chavez S J, Yao J, Fleming T P, Gillanders W E, Wang L V. Photoacoustic microscopy of tyrosinase reporter gene in vivo. J Biomed Optics 2011; 16:080503. PMID: 21895303, PMCID: PMC3162617, DOI: 10.1117/1.3606568
  • 35. Paproski R J, Forbrich A E, Wachowicz K, Htt M M, Zemp R J. Tyrosinase as a dual reporter gene for both photoacoustic and magnetic resonance imaging. Biomed Opt Express 2011; 2:771-780. PMID: 21483602, PMCID: PMC3072120, DOI: 10.1364/BOE.2.000771
  • 36. Qin C, Cheng K, Chen K, Hu X, Liu Y, Lan X, Zhang Y, Liu H, Xu Y, Bu L, Su X, Zhu X, Meng S, Cheng Z. Tyrosinase as a multifunctional reporter gene for Photoacoustic/MRI/PET triple modality molecular imaging. Sci Rep 2013; 3:1490. PMID: 23508226, PMCID: PMC3603217, DOI: 10.1038/srep01490
  • 37. Stritzker J, Kirscher L, Scadeng M, Deliolanis N C, Morscher S, Symvoulidis P, Schaefer K, Zhang Q, Buckel L, Hess M, Donat U, Bradley W G, Ntziachristos V, Szalay A A. Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer. Proc Nat Acad Sci USA 2013; 110:3316-3320. PMID: 23401518, PMCID: PMC3587225, DOI: 10.1073/pnas.1216916110
  • 38. Paproski R J, Heinmiller A, Wachowicz K, Zemp R J. Multi-wavelength photoacoustic imaging of inducible tyrosinase reporter gene expression in xenograft tumors. Sci Rep 2014; 4:5329. PMID: 24936769, PMCID: PMC4060505, DOI: 10.1038/srep05329
  • 39. Feng H, Xia X, Li C, Song Y, Zin C, Zhang Y, Lan X. TYR as a multifunctional reporter gene regulated by the Tet-on system for multimodality imaging: an in vitro study. Sci Rep 2015; 5:15502. PMID: 26483258, PMCID: PMC4611178, DOI: 10.1038/srep15502
  • 40. Paproski R J, Li Y, Barber Q, Lewis J D, Campbell R E, Zemp R. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli. J Biomed Optics 2015; 20:106008. PMID: 26502231, no PMCID, DOI: 10.1117/1.JBO.20.10.106008
  • 41. Jathoul A P, Laufer J, Ogunlade O, Treeby B, Cox B, Zhang E, Johnson P, Pizzey A R, Philip B, Marafioti T, Lythgoe M F, Bedley R B, Pule M A, Beard P. Deep in vivo photoacoustic imaging of mammalian tissues using a tyrosine-based genetic reporter. Nature Photonics 2015; 9:239-246. No PMID, no PMCID, DOI: 10.1038/nphoton.2015.22
  • 42. Brunker J, Yao J, LAufer J, Bohndiek S E. Photoacoustic imaging using genetically encoded reporters: a review. J Biomed Optics 2017; 22:070901. PMID: 28717818, no PMCID, DOI: 10.1117/1.JBO.22.7.070901
  • 43. Levi J, Kothapalli S R, Ma T J, Hartman K, Khuri-Yakub B T, Gambhir S S. Design, synthesis and imaging of an activatable photoacoustic probe. J Am Chem Soc 2010; 132:11264-11269. PMID: 20698693, PMCID: PMC2922742, DOI: 10.1021/ja104000a
  • 44. Xia X, Yang M, Oetj en LK, Zhang Y, Li O, Chen J, Xia Y. An enzyme-sensitive probe for photoacoustic imaging and fluorescence detection of protease activity. Nanoscale 2011; 3:950-953. PMID: 21225037, no PMCID, DOI: 10.1039/c0nr00874e.
  • 45. Zha Z, Zhang S, Deng Z, Li Y, Li C, Dai Z. Enzyme-responsive copper sulphide nanoparticles for combined photoacoustic imaging, tumor-selective chemotherapy and photothermal therapy. Chem Comm 2013; 49:3455-457. PMID: 23507786, no PMCID, DOI: 10.1039/c3cc40608c
  • 46. Dragulescu-Andrasi A, Kothapalli S R, Tikhomirov G A, Rao J, Gambhir S S. Activatable oligomerizable imaging agents for photoacoustic imaging of furin-like activity in living subjects. J Am Chem Soc 2013; 135:11015-11022. PMID: 23859847, PMCID: PMC3771329, DOI: 10.1021/ja4010078
  • 47. Yang K, Zhu L, Nie L, Sun X, Cheng L, Wu C, Niu G, Chen X, Liu Z. Visualization of protease activity in vivo using an activatable photoacoustic imaging probe based on CuS nanoparticles. Theranostics 2014; 4:134-141. PMID: 24465271, PMCID: PMC3900798, DOI: 10.7150/thno.7217
  • 48. Hirasawa T, Iwatate R J, Kamiya M, Okawa S, Urano Y, Ishihara M. Multispectral photoacouostic imaging of tumours in mice injected with an enzyme-activatable photoacoustic probe. J Optics 2017; 19:014002. PMID: 29736563, no PMCID, DOI: 10.1007/s11307-018-1203-1
  • 49. Grootendorst D J, Jose J, Wouters M W, van Boven H, Van der Hage J, Van Leeuwen D G, Steenbergen W, Manohar S, Ruers T J M. First experiences of photoacoustic imaging for detection of melanoma metastases in resected human lymph nodes. Lasers Surg Med 2012; 44:541-549. PMID: 22886491, no PMCID, DOI: 10.1002/1sm.22058
  • 50. Zhou Y, Li G, Zhu L, Li C, Cornelius L A, Wang L V. Handheld photoacoustic probe to detect both melanoma depth and volume at high speed in vivo. J Biophotonics 2015; 8:961-967. PMID: 25676898, PMCID: PMC4530093, DOI: 10.1002/jbio.201400143
  • 51. Stoffels I, Morscher S, Helfrich I, Hillen U, Leyh J, Burton N C, Sardella T C P, Claussen J, Poeppel T D, Bachmann H S, Roesch A, Briewank K, Schadendorf D, Gunzer M, Klode J. Metastatic status of sentinel lymph nodes in melanoma determined noninvasively with multispectral optoacoustic imaging. Sci Trans Med 2015; 7:317ra199. PMID: 26659573, no PMCID, DOI: 10.1126/scitranslmed.aad1278
  • 52. Schwarz M, Buehler A, Aguirre J, Ntziachristos V. Three-dimensional multispectral optoacoustic mesoscopy reveals melanin and blood oxygenation in human skin in vivo. J Biophotonics 2016; 9:55-60. PMID: 26530688, no PMCID, DOI: 10.1002/jbio.201500247
  • 53. Lavaud J, Henry M, Coll J L, Josserand V. Exploration of melanoma metastases in mice brains using endogenous contrast photoacoustic imaging. Int J Pharmaceutics 2017; 532:704-709. PMID: 28847669, no PMCID, DOI: 10.1016/j.ijpharm.2017.08.104
  • 54. Shah A, Delgado-Goni T, Casals Galobart T, Wantuch S, Jamin Y, Leach M O, Robinson S P, Bamber J, Beloueche-Babari M. Detecting human melanoma cell re-differentiation following BRAF or heat shock protein 90 inhibition using photoacoustic and magnetic resonance imaging. Sci Rep 2017; 7:8215. PMID: 28811486, PMCID: PMC5557970, DOI: 10.1038/s41598-017-07864-8
  • 55. Shabat D. Self-immolative dendrimers as novel drug delivery platforms. J Poly Sci Part A 2006; 44:1569-1578. No PMID, no PMCID, DOI: 10.1002/pola.21258
  • 56. Blencowe C A, Russell A T, Greco F, Hayes W, Thornthwaite D W. Self-immolative linkers in polymeric delivery systems. Polymer Chem 2011; 2:773-790. No PMID, no PMCID, DOI: 10.1039/COPY00324G
  • 57. Rosen B M, Wilson C J, Wilson D A, Peterca M, Imam M R, Percec V. Dendron-mediated self-assembly, disassembly, and self-organization of complex systems. Chem Rev 2009; 109:6275-6540. PMID: 19877614, no PMCID, DOI: 10.1021/cr900157q
  • 58. Li Y, Sheth V R, Liu G, Pagel M D. A self-calibrating PARACEST MM contrast agent that detects esterase enzyme activity. Contrast Media Molec Imaging 2011; 6:219-228. PMID 21861282, PMCID: PMC4879975, DOI: 10.1002/cmmi.421
  • 59. Zhang R, Fan Q, Yang M, Cheng K, Lu X, Zhang L, Huang W, Cheng Z. Engineering melanin nanoparticles as an efficient drug-delivery system for imaging-guided chemotherapy. 2015; 27:5063-5069. PMID: 26222210, no PMCID, DOI: 10.1002/adma.201502201
  • 60. Liopo A, Su R, Orafesky A A, Melanin nanoparticles as a novel contrast agent for optoacoustic tomography. Photoacoustics 2015; 3:35-43. No PMID, no PMCID, DOI: 10.1016/j.pacs.2015.02.001
  • 61. Fan Q, Cheng K, Hu X, Ma X, Zhang R, Yang M, Lu X, Xing L, Huuang W, Gambhir S S, Cheng Z. Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging. J Am Chem Soc 2014; 136:15185-15194. PMID: 25292385, PMCID: PMC4227813, DOI: 10.1021/ja505412p
  • 62. Longo D L, Stefania R, Aime S, Oraevsky A. Melanin-based contrast agents for biomedical optoacoustic imaging and theranostic applications. Int J Molec Sci 2017; 18:1719. PMID: 28783106, PMCID: PMC5578109, DOI: 10.3390/ijms18081719
  • 63. Ito S, Inoue S, Yamamoto Y, Fujita K. Synthesis and antitumor activity of cysteinyl-3,4-dihydroxyphenylalanines and related compounds. J Med Chem 1981; 24:673-677. PMID: 6788955, no PMCID, no DOI
  • 64. Chen C, Zhu Y F, Wilcoxen K. An improved synthesis of selectivity protected L-dopa derivatives from L-tyrosine. J Org Chem 2000; 65:2574-2576. No PMID, no PMCID, DOI: 10.1021/jo9913661
  • 65. He G, Lu C, Zhao Y, Nack W A, Chen G. Improved protocol for indoline synthesis via palladium-catalyzed intramolecular C(sp2)-H amination. Org Lett 2012; 14:2944-2947. PMID: 22670815, no PMCID, no DOI.
  • 66. Fernandez-Cuervo G, Tucker K A, Malm S W, Jones K M, Pagel M D. Diamagnetic imaging agents with a modular chemical design for quantitative detection of 3-galactosidase and β-glucuronidase activities with catalyCEST MM. Bioconjug Chem 2016, 27:2549-2557. PMID: 27657647, no PMCID, DOI: 10.1021/acs.bioconjchem.6b00482.
  • 67. Hingorani D V, Montano L A, Randtke E A, Lee Y S, Cardenas-Rodriguez J, Pagel M D. A single diamagnetic catalyCEST Mill contrast agent that detects cathepsin B enzyme activity by using a ratio of two CEST signals. Contrast Media Molec Imaging 2016; 11:130-138. PMID: 26633584, PMCID: PMC4882611, DOI: 10.1002/cmmi.1672
  • 68. Yoo B, Pagel M D. Peptidyl molecular imaging contrast agents using a new solid phase peptide synthesis approach. Bioconj Chem 2007; 18:903-911. PMID 17330953, PMCID: PMC2584118, DOI: 10.1021/bc060250q
  • 69. Yoo B, Pagel M D. A facile synthesis of α-amino-DOTA as a versatile molecular imaging probe. Tet Lett 2006; 47:7327-7330. No PMID, no PMCID, DOI: 10.1016/j.tetlet.2006.08.019
  • 70. Liu G, Li Y, Pagel M D. Design and characterization of new irreversible responsive PARACEST MRI contrast agent that detects nitric oxide. Magn Reson Med 2007; 58:1249-1256. PMID 18046705, no PMCID, DOI: 10.1002/mrm.21428
  • 71. Yoo B, Sheth V, Pagel M D. An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis. Tet Lett 2009; 50:4459-4462. PMID 20161272, PMCID: PMC2702766, DOI: 10.1016/j.tetlet.2009.05.061
  • 72. Ali M M, Yoo B, Pagel M D. Tracking the relative in vivo pharmacokinetics of nanoparticles with PARACEST MM. Molec Pharmaceutics 2009; 6:1409-1416. PMID 19298054, PMCID: PMC4216567, DOI: 10.1021/mp900040u
  • 73. Hingorani D V, Randtke E A, Pagel M D. A catalyCEST MM contrast agent that detects the enzyme-catalyzed creation of a covalent bond. J Am Chem Soc 2013; 135:6396-6398. PMID 23601132, PMCID: PMC3667154, DOI: 10.1021/j a400254e.
  • 74. Yoo B, Sheth V R, Howison C M, Douglas M J K, Pineda C T, Maine E A, Baker A F, Pagel M D. Detection of in vivo enzyme activity with catalyCEST MM. Mag Reson Med 2014; 71:1221-1230. PMID 23640714, PMCID: PMC3742626, DOI: 10.1002/mrm.24763.
  • 75. Fernandez-Cuervo G, Sinharay S, Pagel M D. A catalyCEST MRI contrast agent that can simultaneously detect two enzyme activities. ChemBioChem, 2016; 17(5):383-387. PMID: 26693680, PMCID: PMC4814164, DOI: 10.1002/cbic.201500586.
  • 76. Sinharay S, Fernandez-Cuervo G, Acfalle J P, Pagel M D. Detection of sulfatase enzyme activity with a catalyCEST Mill contrast agent. Chem Euro J 2016; 22:6491-6495. PMID: 26956002, PMCID: PMC4877021, DOI: 10.1002/chem.201600685
  • 77. Daryaei I, Randtke E A, Pagel M D. A biomarker-responsive T2ex MRI contrast agent. Magn Reson Med 2017; 77:1665-1670. PMID: 27090199, PMCID: PMC5071101, DOI: 10.1002/mrm.26250
  • 78. Sinharay S, Randtke E A, Jones K M, Howison C M, Chambers S K, Kobayashi H, Pagel M D. Noninvasive detection of enzyme activity in tumor models of human ovarian cancer using catalyCEST MM. Magn Reson Med 2017; 77:2005-2014. PMID: 27221386, PMCID: PMC5123981, DOI: 10.1002/mrm.26278
  • 79. Daryaei I, Jones K M, Pagel M D. Detection of DT-diaphorase enzyme with a paraCEST MRI contrast agent. Chem Euro J 2017; 23:6514-6517. PMID: 28370655, no PMCID, DOI: 10.1002/chem.201700721.
  • 80. Sinharay S, Howison C M, Baker A F, Pagel M D. Detecting in vivo urokinase Plasminogen Activator activity with a catalyCEST MM contrast agent. NMR Biomed 2017, 30(7):e3721. PMID: 28370884, PMCID: PMC5704996, DOI: 10.1002/nbm.3721
  • 81. Sinharay S, Randtke E A, Howison C M, Ignatenko N A, Pagel M D. Detection of enzyme activity and inhibition during studies in solution, in vitro and in vivo with catalyCEST MM. Molec Imaging Biol 2018; 20:240-248. PMID: 28726131, no PMCID, DOI: 10.1007/s11307-017-1092-8
  • 82. Shamis M, Lode H N, Shabat D. Bioactivation of self-immolative dendritic prodrugs by catalytic antibody 38C2. J Am Chem Soc 2004; 126:1726-1731. PMID: 14871103, no PMCID, DOI: 10.1021/ja039052p
  • 83. Hupple C W, Morscher S, Burton N C, Pagel M D., McNally L R, Cardenas-Rodriguez J. A light-fluence-independent method for the quantitative analysis of dynamic contrast-enhanced multispectral optoacoustic tomography (DCE MSOT). Photoacoustics 2018; 10:54-64. No PMID, no PMCID, DOI: 10.1016/j.pacs.2018.04.003
  • 84. Yoo B, Pagel M D. A PARACEST MM contrast agent to detect enzyme activity. J Am Chem Soc 2006; 128:14032-14033. PMID: 17061878, no PMCID, DOI: 10.1021/ja063874f
  • 85. Yoo B, Raam M, Rosenblum R, Pagel M D. Enzyme-responsive PARACEST MRI contrast agents: A new biomedical imaging approach for studies of the proteasome. Contrast Media Molec Imaging 2007; 2:189-198. PMID: 17712869, no PMCID, DOI: 10.1002/cmmi.145
  • 86. Jones K M, Pagel M D., Cárdenas-Rodríguez J. Linearization improves the repeatability of quantitative dynamic contrast enhanced MRI. Magn Reson Imaging, 2018; 47:16-24. PMID: 29155024, PMCID: PMC5828901, DOI: 10.1016/j.mri.2017.11.002
  • 87. Flurkey W H, Inlow J K. Use of mushroom tyrosinase to introduce Michaelis-Menten enzyme kinetics to biochemistry students. Biochem Mol Biol Educ 2017; 45:270-276. PMID: 28509370, no PMCID, DOI: 10.1002/bmb.21029.
  • 88. Selvarajan E, Mohanasrinivasan V. Kinetic studies on exploring lactose hydrolysis potential of □ galctosidase extracted from Lactobacillus plantarum HF571129. J Food Sci Technol 2015; 52:6206-6217. PMID: 26396367, PMCID: PMC4573140, DOI: 10.1007/s13197-015-1729-z
  • 89. Farnleitner A H, Hocke L, Beiwl C, Kavka G G, Mach R L. Hydrolysis of 4-methylumbelliferyl-beta-D-glucuronidase in differing sample fractions of river waters and its implication for the detection of fecal pollution. Water Res 2002; 36:975-981. PMID: 11848369, no PMCID, no DOI.
  • 90. De Bruyne C K, Yde M. Binding of alkyl I-thio-□-D-galactopyranosides to −□-D-galactosidase from E. coli. Carbohydrate Res 1977; 56:153-164. PMID: 18283, no PMCID, no DOI.
  • 91. Wallace B D, Wang H, Lane K T, Scott J E, ORans J, Koo J S, Venkatesh M, Jobin C, yeh LA, Mani S, Redinbo M R. Alleviating cancer drug toxicity by inhibiting a bacterial enzyme. Science 2010; 330:831-835. PMID 21051639; PMCID: PMC3110694, DOI: 10.1126/science.1191175
  • 92. Randtke E A, Chen L Q, Corrales L R, Pagel M D. The Hanes-Woolf Linear QUESP method improves the measurements of fast chemical exchange rates with CEST MRI Magn Reson Med 2014; 71:1603-1612. PMID 23780911, PMCID: PMC3784632, DOI: 10.1002/mrm.24792.
  • 93. Fernandez-Cuervo Velasco G. Design, synthesis and application of catalyCEST Mill agents for enzyme detection. PhD Thesis, University of Arizona, 2017. No PIMD, no PMCID, no DOI.
  • 94. Kobes J E, Daryaei I, Howison C M, Bontrager J G, Siriani R S, Meuillet E M, Pagel M D. Improved treatment of pancreatic cancer with drug delivery nanoparticles loaded with a novel AKT/PDK1 inhibitor. Pancreas 2016; 45:1158-1166. PMID: 26918875, PMCID: PMC4983222, DOI: 10.1097/MPA.0000000000000607.
  • 95. Chen L Q, Randtke E A, Jones K M, Moon B F, Howison C M, Pagel M D. Evaluations of tumor acidosis within in vivo tumor models using parametric maps generated with acidoCEST MRL Mol Imaging Biol 2015; 17:488-496. PMID: 25622809, PMCID: PMC4880367, DOI: 10.1007/s11307-014-0816-2.
  • 96. Miller L R, Marks C, Becker J B, Hurn P D, Chen W J, Woodruff T, McCarthy M M Sohrabji F, Schiebinger L, Wetherington C L, Makris S, Arnold A P, Einstein G, Miller V M, Sandberg K, Maier S, Cornelison T L, Clayton J A. Considering sex as a biological variable in preclinical research. FASEB J, 2017; 31:29-34. PMID: 27682203, no PMCID, DOI: 10.1096/fj.201600781R

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. A composition, comprising:

a compound, comprising: a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, and a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or the indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity.

2. The composition of claim 1, wherein the melanin precursor is selected from the group consisting of leucodopachrome, decarboxylated leucodopachrome, and cysteinyldopa.

3. The composition of claim 1, wherein the indirect link comprises ap-hydroxybenzyl moiety.

4. The composition of claim 2, wherein the compound is selected from the group consisting of Agent 1, Agent 2, Agent 3, and Agent 6:

wherein n is greater than or equal to 1 and each R is any organic moiety.

5. The composition of claim 1, further comprising:

a pharmaceutically-acceptable carrier.

6. A method, comprising:

administering, to a patient suffering from a tumor, a composition comprising a compound comprising a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, and a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or the indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity; and
surgically resecting the tumor, wherein the surgical resection is guided at least in part by contrast imparted by the melanin, wherein the melanin is spontaneously synthesized in the tumor after administering the composition.

7. The method of claim 6, wherein the tumor is a solid tumor comprising cells that express high levels of protease.

8. The method of claim 7, wherein the tumor is of a cancer selected from the group consisting of breast cancer, head-and-neck cancer, pancreatic cancer, ovarian cancer, and thyroid cancer.

9. The method of claim 6, wherein the melanin precursor in the compound in the administered composition is selected from the group consisting of leucodopachrome, decarboxylated leucodopachrome, and cysteinyldopa.

10. The method of claim 6, wherein the indirect link between the melanin precursor and the peptide in the compound in the administered composition comprises a p-hydroxybenzyl moiety.

11. The method of claim 10, wherein the compound in the administered composition is selected from the group consisting of Agent 1, Agent 2, Agent 3, and Agent 6:

wherein n is greater than or equal to 1 and each R may be any organic moiety.

12. The method of claim 6, further comprising:

administering, to the patient, a cancer treatment modality other than surgical resection.

13. The method of claim 12, wherein the cancer treatment modality other than surgical resection is selected from the group consisting of radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitor therapy, oncolytic virus therapy, peptide therapy, thermal therapy, and two or more thereof.

14. A method, comprising:

administering, to a patient suffering from a tumor, a composition comprising a compound comprising a melanin precursor that spontaneously synthesizes a melanin when in free form in vivo, and a peptide directly covalently bonded to or indirectly linked to the melanin precursor, wherein the direct covalent bond or the indirect link of the peptide to the melanin precursor blocks spontaneous synthesis of the melanin in vivo in the absence of protease activity; and
thermally ablating the tumor, wherein the thermal ablating targets cells containing the melanin, wherein the melanin is spontaneously synthesized in the tumor after administering the composition.

15. The method of claim 14, wherein the tumor is a solid tumor comprising cells that express high levels of protease.

16. The method of claim 14, wherein the melanin precursor in the compound in the administered composition is selected from the group consisting of leucodopachrome, decarboxylated leucodopachrome, and cysteinyldopa.

17. The method of claim 14, wherein the indirect link between the melanin precursor and the peptide in the compound in the administered composition comprises a p-hydroxybenzyl moiety.

18. The method of claim 17, wherein the compound in the administered composition is selected from the group consisting of Agent 1, Agent 2, Agent 3, and Agent 6:

wherein n is greater than or equal to 1 and each R may be any organic moiety.

19. The method of claim 14, further comprising:

administering, to the patient, a cancer treatment modality other than thermal ablation.

20. The method of claim 19, wherein the cancer treatment modality other than thermal ablation is selected from the group consisting of radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitor therapy, oncolytic virus therapy, peptide therapy, cryotherapy, and two or more thereof.

Patent History
Publication number: 20230248849
Type: Application
Filed: Jul 26, 2021
Publication Date: Aug 10, 2023
Applicant: BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM (Austin, TX)
Inventor: Mark D. PAGEL (Houston, TX)
Application Number: 18/004,827
Classifications
International Classification: A61K 49/00 (20060101); A61K 45/06 (20060101); A61P 35/00 (20060101);